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Wei T, Liu H, Chu B, Blasco P, Liu Z, Tian R, Li DX, Li X. Phosphorylation-regulated HMGA1a-P53 interaction unveils the function of HMGA1a acidic tail phosphorylations via synthetic proteins. Cell Chem Biol 2021; 28:722-732.e8. [PMID: 33545070 DOI: 10.1016/j.chembiol.2021.01.007] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2020] [Revised: 11/13/2020] [Accepted: 01/06/2021] [Indexed: 01/10/2023]
Abstract
As a typical member of intrinsically disordered proteins (IDPs), HMGA1a carries many post-translational modifications (PTMs). To study the undefined function of acidic tail phosphorylations, seven HMGA1a proteins with site-specific modification(s) were chemically synthesized via Ser/Thr ligation. We found that the phosphorylations significantly inhibit HMGA1a-P53 interaction and the phosphorylations can induce conformational change of HMGA1a from an "open state" to a "close state." Notably, the positively charged lysine-arginine (KR) clusters are responsible for modulating HMGA1a conformation via electrostatic interaction with the phosphorylated acidic tail. Finally, we used a synthetic protein-affinity purification mass spectrometry (SP-AP-MS) methodology to profile the specific interactors, which further supported the function of HMGA1a phosphorylation. Collectively, this study highlights a mechanism for regulating IDPs' conformation and function by phosphorylation of non-protein-binding domain and showcases that the protein chemical synthesis in combination with mass spectrometry can serve as an efficient tool to study the IDPs' PTMs.
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Affiliation(s)
- Tongyao Wei
- Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, P. R. China
| | - Heng Liu
- Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, P. R. China
| | - Bizhu Chu
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, P. R. China
| | - Pilar Blasco
- Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, P. R. China
| | - Zheng Liu
- Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, P. R. China
| | - Ruijun Tian
- Department of Chemistry, Southern University of Science and Technology, Shenzhen, P. R. China
| | - David Xiang Li
- Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, P. R. China
| | - Xuechen Li
- Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, P. R. China.
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2
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Murawska GM, Poloni C, Simeth NA, Szymanski W, Feringa BL. Comparative Study of Photoswitchable Zinc-Finger Domain and AT-Hook Motif for Light-Controlled Peptide-DNA Binding. Chemistry 2019; 25:4965-4973. [PMID: 30735272 DOI: 10.1002/chem.201900090] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2019] [Indexed: 12/20/2022]
Abstract
DNA-peptide interactions are involved in key life processes, including DNA recognition, replication, transcription, repair, organization, and modification. Development of tools that can influence DNA-peptide binding non-invasively with high spatiotemporal precision could aid in determining its role in cells and tissues. Here, the design, synthesis, and study of photocontrolled tools for sequence-specific small peptide-DNA major and minor groove interactions are reported, shedding light on DNA binding by transcriptionally active peptides. In particular, photoswitchable moieties were implemented in the peptide backbone or turn region. In each case, DNA binding was affected by photochemical isomerization, as determined in fluorescent displacement assays on model DNA strands, which provides promising tools for DNA modulation.
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Affiliation(s)
- Gosia M Murawska
- Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747, AG, Groningen, The Netherlands
| | - Claudia Poloni
- Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747, AG, Groningen, The Netherlands
| | - Nadja A Simeth
- Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747, AG, Groningen, The Netherlands
| | - Wiktor Szymanski
- Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747, AG, Groningen, The Netherlands.,Department of Radiology, University of Groningen, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands
| | - Ben L Feringa
- Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747, AG, Groningen, The Netherlands
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3
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Narayan S, Ramisetti S, Jaiswal AS, Law BK, Singh-Pillay A, Singh P, Amin S, Sharma AK. ASR352, A potent anticancer agent: Synthesis, preliminary SAR, and biological activities against colorectal cancer bulk, 5-fluorouracil/oxaliplatin resistant and stem cells. Eur J Med Chem 2019; 161:456-467. [PMID: 30384048 PMCID: PMC7115410 DOI: 10.1016/j.ejmech.2018.10.052] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2018] [Revised: 10/07/2018] [Accepted: 10/22/2018] [Indexed: 12/21/2022]
Abstract
Despite new agent development and short-term benefits in patients with colorectal cancer (CRC), metastatic CRC cure rates have not improved due to high rates of 5-fluorouracil (5-FU)/leucovorin/oxaliplatin (FOLFOX)-resistance and a clinical therapeutic plateau. At the same time, this treatment regime leads to significant toxicity, cost, and patient inconvenience. Drug-resistance is linked to CRC stem cells, which are associated with the epidermal-to-mesenchymal transition (EMT) pathway. Thus, to optimally treat CRC, a therapy that can target the cell survival and EMT pathways in both CRC bulk and stem cell populations is critical. We recently identified a novel small molecule NSC30049 (7a) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC bulk, FOLFOX-resistant, and CRC stem cells both in vitro and in vivo models. In the present study, we report the synthesis and anti-CRC evaluation of several stable and effective 7a analogs. ASR352 (7b) was identified as one of the equipotent 7a analogs that inhibited the growth of CRC bulk cells, sensitized FOLFOX-resistant cells, and reduced the sphere formation capacity of CRC stem cells. It appears that the complex mechanism of cytotoxicity for 7b includes abrogation of 5-FU-induced the S phase, reduction of the phosphorylation of Chk1 at S317P, S345P and S296P, increased γH2AX staining, activation of caspase 3/PARP1 cleavage, and enhancement of Bax/Bcl2 ratio. Further 7b-mediated reduced phosphorylation of Chk1 was an indirect effect, since it did not inhibit Chk1 activity in an in vitro kinase assay. Our findings suggest that 7b as a single agent, or in combination with 5-FU can be developed as a therapeutic agent in CRC bulk, FOLFOX-resistant, and CRC stem cell populations for unmanageable metastatic CRC conditions.
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Affiliation(s)
- Satya Narayan
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL, 32610, USA.
| | - Srinivasa Ramisetti
- Department of Pharmacology, Penn State University College of Medicine, Penn State Cancer Institute, Hershey, PA, 17033, USA
| | - Aruna S Jaiswal
- Department of Hematology and Oncology, University of Florida, Gainesville, FL, 32610, USA
| | - Brian K Law
- Department of Pharmacology and Experimental Therapeutics, University of Florida, Gainesville, FL, 32610, USA
| | - Ashona Singh-Pillay
- School of Chemistry and Physics, University of Kwa-Zulu Natal (UKZN), Westville Campus, Durban, 4000, South Africa
| | - Parvesh Singh
- School of Chemistry and Physics, University of Kwa-Zulu Natal (UKZN), Westville Campus, Durban, 4000, South Africa
| | - Shantu Amin
- Department of Pharmacology, Penn State University College of Medicine, Penn State Cancer Institute, Hershey, PA, 17033, USA
| | - Arun K Sharma
- Department of Pharmacology, Penn State University College of Medicine, Penn State Cancer Institute, Hershey, PA, 17033, USA.
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4
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Emerick B, Schleiniger G, Boman BM. Multi-scale modeling of APC and [Formula: see text]-catenin regulation in the human colonic crypt. J Math Biol 2018; 76:1797-1830. [PMID: 29302705 DOI: 10.1007/s00285-017-1204-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2017] [Revised: 12/22/2017] [Indexed: 10/18/2022]
Abstract
Stem cell renewal and differentiation in the human colonic crypt are linked to the [Formula: see text]-catenin pathway. The spatial balance of Wnt factors in proliferative cells within the crypt maintain an appropriate level of cellular reproduction needed for normal crypt homeostasis. Mutational events at the gene level are responsible for deregulating the balance of Wnt factors along the crypt, causing an overpopulation of proliferative cells, a loss of structure of the crypt domain, and the initiation of colorectal carcinomas. We formulate a PDE model describing cell movement and reproduction in a static crypt domain. We consider a single cell population whose proliferative capabilities are determined by stemness, a quantity defined by intracellular levels of adenomatous polyposis coli (APC) scaffold protein and [Formula: see text]-catenin. We fit APC regulation parameters to biological data that describe normal protein gradients in the crypt. We also fit cell movement and protein flux parameters to normal crypt characteristics such as renewal time, total cell count, and proportion of proliferating cells. The model is used to investigate abnormal crypt dynamics when subjected to a diminished APC gradient, a scenario synonymous to mutations in the APC gene. We find that a 25% decrease in APC synthesis leads to a fraction of 0.88 proliferative, which is reflective of normal-appearing FAP crypts. A 50% drop in APC activity yields a fully proliferative crypt showing a doubling of the level of stemness, which characterizes the initial stages of colorectal cancer development. A sensitivity analysis of APC regulation parameters shows the perturbation of factors that is required to restore crypt dynamics to normal in the case of APC mutations.
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Affiliation(s)
- Brooks Emerick
- Department of Mathematics, Kutztown University, Kutztown, PA, 19530, USA.
| | - Gilberto Schleiniger
- Department of Mathematical Sciences, University of Delaware, Newark, DE, 19711, USA
| | - Bruce M Boman
- Department of Biological Sciences, University of Delaware, Newark, DE, 19711, USA.,Center for Translational Cancer Research, Helen F. Graham Cancer Center and Research Institute, Newark, DE, 19713, USA
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5
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Narayan S, Jaiswal AS, Sharma R, Nawab A, Duckworth LV, Law BK, Zajac-Kaye M, George TJ, Sharma J, Sharma AK, Hromas RA. NSC30049 inhibits Chk1 pathway in 5-FU-resistant CRC bulk and stem cell populations. Oncotarget 2017; 8:57246-57264. [PMID: 28915668 PMCID: PMC5593639 DOI: 10.18632/oncotarget.19778] [Citation(s) in RCA: 12] [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: 04/28/2017] [Accepted: 07/20/2017] [Indexed: 01/20/2023] Open
Abstract
The 5-fluorouracil (5-FU) treatment induces DNA damage and stalling of DNA replication forks. These stalled replication forks then collapse to form one sided double-strand breaks, leading to apoptosis. However, colorectal cancer (CRC) stem cells rapidly repair the stalled/collapsed replication forks and overcome treatment effects. Recent evidence suggests a critical role of checkpoint kinase 1 (Chk1) in preventing the replicative stress. Therefore, Chk1 kinase has been a target for developing mono or combination therapeutic agents. In the present study, we have identified a novel orphan molecule NSC30049 (NSC49L) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC heterogeneous bulk and FOLFOX-resistant cell lines in culture with minimal effect on normal colonic epithelial cells. It also inhibits the sphere forming activity of CRC stem cells, and decreases the expression levels of mRNAs of CRC stem cell marker genes. Results showed that NSC49L induces 5-FU-mediated S-phase cell cycle arrest due to increased load of DNA damage and increased γ-H2AX staining as a mechanism of cytotoxicity. The pharmacokinetic analysis showed a higher bioavailability of this compound, however, with a short plasma half-life. The drug is highly tolerated by animals with no pathological aberrations. Furthermore, NSC49L showed very potent activity in a HDTX model of CRC stem cell tumors either alone or in combination with 5-FU. Thus, NSC49L as a single agent or combined with 5-FU can be developed as a therapeutic agent by targeting the Chk1 pathway in 5-FU-resistant CRC heterogeneous bulk and CRC stem cell populations.
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Affiliation(s)
- Satya Narayan
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA
| | - Aruna S. Jaiswal
- Department of Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Ritika Sharma
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA
| | - Akbar Nawab
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA
| | - Lizette Vila Duckworth
- Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Brian K. Law
- Department of Pharmacology and Experimental Therapeutics, University of Florida, Gainesville, FL 32610, USA
| | - Maria Zajac-Kaye
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA
| | - Thomas J. George
- Department of Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Jay Sharma
- Celprogen, Inc., Torrance, CA 90503, USA
| | - Arun K. Sharma
- Department of Pharmacology, Penn State Cancer Institute, Penn State College of Medicine, Hershey, PA 17033, USA
| | - Robert A. Hromas
- Department of Medicine, University of Florida, Gainesville, FL 32610, USA
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Abstract
Growth hormone (GH) excess in acromegaly is associated with increased precancerous colon polyps and soft tissue adenomas, whereas short-stature humans harboring an inactivating GH receptor mutation do not develop cancer. We show that locally expressed colon GH is abundant in conditions predisposing to colon cancer and in colon adenocarcinoma-associated stromal fibroblasts. Administration of a GH receptor (GHR) blocker in acromegaly patients induced colon p53 and adenomatous polyposis coli (APC), reversing progrowth GH signals. p53 was also induced in skin fibroblasts derived from short-statured humans with mutant GHR. GH-deficient prophet of pituitary-specific positive transcription factor 1 (Prop1)(-/-) mice exhibited induced colon p53 levels, and cross-breeding them with Apc(min+/-) mice that normally develop intestinal and colon tumors resulted in GH-deficient double mutants with markedly decreased tumor number and size. We also demonstrate that GH suppresses p53 and reduces apoptosis in human colon cell lines as well as in induced human pluripotent stem cell-derived intestinal organoids, and confirm in vivo that GH suppresses colon mucosal p53/p21. GH excess leads to decreased colon cell phosphatase and tensin homolog deleted on chromosome 10 (PTEN), increased cell survival with down-regulated APC, nuclear β-catenin accumulation, and increased epithelial-mesenchymal transition factors and colon cell motility. We propose that GH is a molecular component of the "field change" milieu permissive for neoplastic colon growth.
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Kuo TL, Weng CC, Kuo KK, Chen CY, Wu DC, Hung WC, Cheng KH. APC haploinsufficiency coupled with p53 loss sufficiently induces mucinous cystic neoplasms and invasive pancreatic carcinoma in mice. Oncogene 2016; 35:2223-34. [PMID: 26411367 DOI: 10.1038/onc.2015.284] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2014] [Revised: 06/03/2015] [Accepted: 06/22/2015] [Indexed: 12/23/2022]
Abstract
Adenomatous polyposis coli (APC), a tumor-suppressor gene critically involved in familial adenomatous polyposis, is integral in Wnt/β-catenin signaling and is implicated in the development of sporadic tumors of the distal gastrointestinal tract including pancreatic cancer (PC). Here we report for the first time that functional APC is required for the growth and maintenance of pancreatic islets and maturation. Subsequently, a non-Kras mutation-induced premalignancy mouse model was developed; in this model, APC haploinsufficiency coupled with p53 deletion resulted in the development of a distinct type of pancreatic premalignant precursors, mucinous cystic neoplasms (MCNs), exhibiting pathomechanisms identical to those observed in human MCNs, including accumulation of cystic fluid secreted by neoplastic and ovarian-like stromal cells, with 100% penetrance and the presence of hepatic and gastric metastases in >30% of the mice. The major clinical implications of this study suggest targeting the Wnt signaling pathway as a novel strategy for managing MCN.
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Affiliation(s)
- T-L Kuo
- Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
| | - C-C Weng
- Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
| | - K-K Kuo
- Division of Hepatobiliopancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - C-Y Chen
- Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan
- Department of Medical Imaging, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - D-C Wu
- Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan
- Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Division of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - W-C Hung
- National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan
| | - K-H Cheng
- Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
- Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan
- Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan
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8
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Interaction between APC and Fen1 during breast carcinogenesis. DNA Repair (Amst) 2016; 41:54-62. [PMID: 27088617 DOI: 10.1016/j.dnarep.2016.04.003] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 04/06/2016] [Indexed: 02/07/2023]
Abstract
Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER - adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) - promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.
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Ma L, Ma R, Wang Y, Zhu X, Zhang J, Chan HC, Chen X, Zhang W, Chiu SK, Zhu G. Chalcoplatin, a dual-targeting and p53 activator-containing anticancer platinum(IV) prodrug with unique mode of action. Chem Commun (Camb) 2015; 51:6301-4. [PMID: 25644651 DOI: 10.1039/c4cc10409a] [Citation(s) in RCA: 76] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Complexation of cisplatin with a p53 activator as a single anticancer agent resulted in synergistically improved cytotoxicity in p53 wild-type but not p53 null human cancer cells. Mechanistic investigation was carried out on this dual-targeting Pt(IV) prodrug, chalcoplatin. The prodrug effectively entered cancer cells and arrested the cell cycle at the S and G2/M phases, distinctive of that from cisplatin. Chalcoplatin significantly induced p53 activation as well as the subsequent apoptosis pathways. This unique mode of action renders chalcoplatin remarkably cytotoxic and makes this compound among the first examples of a Pt(IV) prodrug that directly interacts with the downstream pathway after the formation of Pt-DNA lesions.
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Affiliation(s)
- Lili Ma
- Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong SAR.
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Poloni C, Szymański W, Hou L, Browne WR, Feringa BL. A Fast, Visible-Light-Sensitive Azobenzene for Bioorthogonal Ligation. Chemistry 2014; 20:946-51. [DOI: 10.1002/chem.201304129] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2013] [Indexed: 12/22/2022]
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Das D, Preet R, Mohapatra P, Satapathy SR, Kundu CN. 1,3-Bis(2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of Resveratrol on 5-fluorouracil sensitive/resistant colon cancer cells. World J Gastroenterol 2013; 19:7374-7388. [PMID: 24259968 PMCID: PMC3831219 DOI: 10.3748/wjg.v19.i42.7374] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/18/2012] [Revised: 03/22/2013] [Accepted: 06/06/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the mechanism of 5-fluorouracil (5-FU) resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment.
METHODS: We established and characterized a 5-FU resistance (5-FU-R) cell line derived from continuous exposure (25 μmol/L) to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells. The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting, respectively, after treatment with Resveratrol (Res) and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU). Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis, respectively. The extent of DNA damage was measured by the Comet assay. We measured the visible changes in the DNA damage/repair cascade by Western blotting.
RESULTS: The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner. Combined application of BCNU and Res caused more apoptosis in 5-FU sensitive cells in comparison to individual treatment. In addition, the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells. We established a 5-FU resistance cell line (5-FU-R) from 5-FU-sensitive HCT-116 (mismatch repair deficient) cells that was not resistant to other chemotherapeutic agents (e.g., BCNU, Res) except 5-FU. The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay. There was no significant cell death noted in 5-FU-R cells in comparison to 5-FU sensitive cells after 5-FU treatment. This resistant cell line overexpressed anti-apoptotic [e.g., AKT, nuclear factor κB, FLICE-like inhibitory protein), DNA repair (e.g., DNA polymerase beta (POL-β), DNA polymerase eta (POLH), protein Flap endonuclease 1 (FEN1), DNA damage-binding protein 2 (DDB2)] and 5-FU-resistance proteins (thymidylate synthase) but under expressed pro-apoptotic proteins (e.g., DAB2, CK1) in comparison to the parental cells. Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells. BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells. Fifty percent cell death were noted in parental cells when 18 μmol/L of Res was associated with fixed concentration (20 μmol/L) of BCNU, but a much lower concentration of Res (8 μmol/L) was needed to achieve the same effect in 5-FU resistant cells. Interestingly, increased levels of adenomatous polyposis coli and decreased levels POL-β, POLH, FEN1 and DDB2 were noted after the same combined treatment in resistant cells.
CONCLUSION: BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance.
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12
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Boman BM, Fields JZ. An APC:WNT Counter-Current-Like Mechanism Regulates Cell Division Along the Human Colonic Crypt Axis: A Mechanism That Explains How APC Mutations Induce Proliferative Abnormalities That Drive Colon Cancer Development. Front Oncol 2013; 3:244. [PMID: 24224156 PMCID: PMC3819610 DOI: 10.3389/fonc.2013.00244] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2013] [Accepted: 09/03/2013] [Indexed: 12/17/2022] Open
Abstract
APC normally down-regulates WNT signaling in human colon, and APC mutations cause proliferative abnormalities in premalignant crypts leading to colon cancer, but the mechanisms are unclear at the level of spatial and functional organization of the crypt. Accordingly, we postulated a counter-current-like mechanism based on gradients of factors (APC;WNT) that regulate colonocyte proliferation along the crypt axis. During crypt renewal, stem cells (SCs) at the crypt bottom generate non-SC daughter cells that proliferate and differentiate while migrating upwards. The APC concentration is low at the crypt bottom and high at the top (where differentiated cells reside). WNT signaling, in contrast, is high at the bottom (where SCs reside) and low at the top. Given that WNT and APC gradients are counter to one another, we hypothesized that a counter-current-like mechanism exists. Since both APC and WNT signaling components (e.g., survivin) are required for mitosis, this mechanism establishes a zone in the lower crypt where conditions are optimal for maximal cell division and mitosis orientation (symmetric versus asymmetric). APC haploinsufficiency diminishes the APC gradient, shifts the proliferative zone upwards, and increases symmetric division, which causes SC overpopulation. In homozygote mutant crypts, these changes are exacerbated. Thus, APC-mutation-induced changes in the counter-current-like mechanism cause expansion of proliferative populations (SCs, rapidly proliferating cells) during tumorigenesis. We propose this mechanism also drives crypt fission, functions in the crypt cycle, and underlies adenoma development. Novel chemoprevention approaches designed to normalize the two gradients and readjust the proliferative zone downwards, might thwart progression of these premalignant changes.
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Affiliation(s)
- Bruce M. Boman
- Center for Translational Cancer Research, Helen F. Graham Cancer Center and Research Institute, University of Delaware, Newark, DE, USA
- Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
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13
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Enhanced 4-hydroxynonenal resistance in KEAP1 silenced human colon cancer cells. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2013; 2013:423965. [PMID: 23766854 PMCID: PMC3674683 DOI: 10.1155/2013/423965] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/09/2013] [Accepted: 04/09/2013] [Indexed: 12/19/2022]
Abstract
Nuclear factor erythroid 2-related factor 2 (NRF2) is the transcription factor that regulates an array of antioxidant/detoxifying genes for cellular defense. The conformational changes of Kelch-like ECH-associated protein 1 (KEAP1), a cytosolic repressor protein of NRF2, by various stimuli result in NRF2 liberation and accumulation in the nucleus. In the present study, we aimed to investigate the effect of KEAP1 knockdown on NRF2 target gene expression and its toxicological implication using human colon cancer cells. The stable KEAP1-knockdown HT29 cells exhibit elevated levels of NRF2 and its target gene expressions. In particular, the mRNA levels of aldo-keto reductases (AKR1C1, 1C2, 1C3, 1B1, and 1B10) were substantially increased in KEAP1 silenced HT29 cells. These differential AKRs expressions appear to contribute to protection against oxidative stress. The KEAP1-knockdown cells were relatively more resistant to hydrogen peroxide (H2O2) and 4-hydroxynonenal (4HNE) compared to the control cells. Accordantly, we observed accumulation of 4HNE protein adducts in H2O2- or 4HNE-treated control cells, whereas KEAP1-knockdown cells did not increase adduct formation. The treatment of KEAP1-silenced cells with AKR1C inhibitor flufenamic acid increased 4HNE-induced cellular toxicity and protein adduct formation. Taken together, these results indicate that AKRs, which are NRF2-dependent highly inducible gene clusters, play a role in NRF2-mediated cytoprotection against lipid peroxide toxicity.
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14
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Jaiswal AS, Panda H, Pampo CA, Siemann DW, Gairola CG, Hromas R, Narayan S. Adenomatous polyposis coli-mediated accumulation of abasic DNA lesions lead to cigarette smoke condensate-induced neoplastic transformation of normal breast epithelial cells. Neoplasia 2013; 15:454-60. [PMID: 23555190 PMCID: PMC3612917 DOI: 10.1593/neo.13176] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2013] [Revised: 02/06/2013] [Accepted: 02/08/2013] [Indexed: 11/18/2022]
Abstract
Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5'-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model.
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Affiliation(s)
- Aruna S Jaiswal
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL
- Department of Medicine, University of Florida, Gainesville, FL
| | - Harekrushna Panda
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL
| | - Christine A Pampo
- Department of Radiation Oncology, University of Florida, Gainesville, FL
| | - Dietmar W Siemann
- Department of Radiation Oncology, University of Florida, Gainesville, FL
| | - C Gary Gairola
- Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY
| | - Robert Hromas
- Department of Medicine, University of Florida, Gainesville, FL
| | - Satya Narayan
- Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL
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15
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Zeineldin M, Neufeld KL. More than two decades of Apc modeling in rodents. Biochim Biophys Acta Rev Cancer 2013; 1836:80-9. [PMID: 23333833 DOI: 10.1016/j.bbcan.2013.01.001] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2012] [Revised: 12/31/2012] [Accepted: 01/03/2013] [Indexed: 02/07/2023]
Abstract
Mutation of tumor suppressor gene adenomatous polyposis coli (APC) is an initiating step in most colon cancers. This review summarizes Apc models in mice and rats, with particular concentration on those most recently developed, phenotypic variation among different models, and genotype/phenotype correlations.
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Affiliation(s)
- Maged Zeineldin
- Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045, USA
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16
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Adenomatous polyposis coli interacts with flap endonuclease 1 to block its nuclear entry and function. Neoplasia 2012; 14:495-508. [PMID: 22787431 DOI: 10.1593/neo.12680] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2012] [Revised: 05/04/2012] [Accepted: 05/07/2012] [Indexed: 11/18/2022] Open
Abstract
In previous studies, we found that adenomatous polyposis coli (APC) blocks the base excision repair (BER) pathway by interacting with 5'-flap endonuclease 1 (Fen1). In this study, we identify the molecular features that contribute to the formation and/or stabilization of the APC/Fen1 complex that determines the extent of BER inhibition, and the subsequent accumulation of DNA damage creates mutagenic lesions leading to transformation susceptibility. We show here that APC binds to the nuclear localization sequence of Fen1 (Lys(365)Lys(366)Lys(367)), which prevents entry of Fen1 into the nucleus and participation in Pol-β-directed long-patch BER. We also show that levels of the APC/Fen1 complex are higher in breast tumors than in the surrounding normal tissues. These studies demonstrate a novel role for APC in the suppression of Fen1 activity in the BER pathway and a new biomarker profile to be explored to identify individuals who may be susceptible to the development of mammary and other tumors.
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17
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Chen S, Xu D, Jiang H, Xi Z, Zhu P, Liu Y. Trans-Platinum/Thiazole Complex Interferes with Sp1 Zinc-Finger Protein. Angew Chem Int Ed Engl 2012. [DOI: 10.1002/ange.201206596] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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18
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Chen S, Xu D, Jiang H, Xi Z, Zhu P, Liu Y. Trans-Platinum/Thiazole Complex Interferes with Sp1 Zinc-Finger Protein. Angew Chem Int Ed Engl 2012; 51:12258-62. [DOI: 10.1002/anie.201206596] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2012] [Revised: 09/17/2012] [Indexed: 11/11/2022]
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19
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Small-molecule inhibitors of DNA damage-repair pathways: an approach to overcome tumor resistance to alkylating anticancer drugs. Future Med Chem 2012; 4:1093-111. [PMID: 22709253 DOI: 10.4155/fmc.12.58] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
A major challenge in the future development of cancer therapeutics is the identification of biological targets and pathways, and the subsequent design of molecules to combat the drug-resistant cells hiding in virtually all cancers. This therapeutic approach is justified based upon the limited advances in cancer cures over the past 30 years, despite the development of many novel chemotherapies and earlier detection, which often fail due to drug resistance. Among the various targets to overcome tumor resistance are the DNA repair systems that can reverse the cytotoxicity of many clinically used DNA-damaging agents. Some progress has already been made but much remains to be done. We explore some components of the DNA-repair process, which are involved in repair of alkylation damage of DNA, as targets for the development of novel and effective molecules designed to improve the efficacy of existing anticancer drugs.
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20
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Zhang B, Xiang S, Zhong Q, Yin Y, Gu L, Deng D. The p16-specific reactivation and inhibition of cell migration through demethylation of CpG islands by engineered transcription factors. Hum Gene Ther 2012; 23:1071-81. [PMID: 22738793 DOI: 10.1089/hum.2012.070] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Methylation of CpG islands inactivates transcription of tumor suppressor genes including p16 (CDKN2A). Inhibitors of DNA methylation and histone deacylation are recognized as useful cancer therapeutic chemicals through reactivation of the expression of methylated genes. However, these inhibitors are not target gene-specific, so that they lead to serious side effects as regular cytotoxic chemotherapy agents. To explore the feasibility of methylated gene-specific reactivation by artificial transcription factors, we engineered a set of Sp1-like seven-finger zinc-finger proteins (7ZFPs) targeted to a 21-bp sequence of the p16 promoter and found that these 7ZFPs could bind specifically to the target p16 promoter probe. Then the p16-specific artificial transcription factors (p16ATFs) were made from these 7ZFPs and the transcription activator VP64. Results showed that transient transfection of some p16ATFs selectively up-regulated the endogenous p16 expression in the p16-active 293T cells. Moreover, the transient transfection of the representative p16ATF-6I specifically reactivated p16 expression in the p16-methylated H1299 and AGS cells pretreated with a nontoxic amount of 5'-aza-deoxycytidine (20 and 80 nM, respectively). In addition, stable transfection of the p16ATF induced demethylation of p16 CpG island and trimethylation of histone H3K4, and inhibited recruitment of DNA methyltransferase 1 and trimethylation of H3K9 and H3K27 in the p16 promoter in H1299 cells without 5'-aza-deoxycytidine pretreatment. Notably, inhibition of cell migration and invasion was observed in these p16-reactivated cells induced by transient and stable p16ATF transfection. These results demonstrate that p16ATF not only specifically reactivates p16 expression through demethylation of CpG islands, but also restores methylated p16 function.
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Affiliation(s)
- Baozhen Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Aetiology, Peking University Cancer Hospital/Institute, Beijing, 100142, China
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21
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Lu W, Jia G, Meng X, Zhao C, Zhang L, Ren Y, Pan H, Ni Y. Beta-catenin mediates the apoptosis induction effect of celastrol in HT29 cells. Life Sci 2012; 91:279-83. [PMID: 22877649 DOI: 10.1016/j.lfs.2012.07.032] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2012] [Revised: 06/23/2012] [Accepted: 07/16/2012] [Indexed: 12/26/2022]
Abstract
AIM We evaluated the apoptosis induction effects of celastrol in human colorectal cancer cell line HT29 in WNT/beta-catenin pathway. MAIN METHODS HT29 cells were treated with various concentrations (10-100μM) for 24h, MTT assay was performed to examine the effect of celastrol on growth inhibition of HT29 cells. Beta-catenin siRNA was used for transfection of cells. Cell apoptosis was detected through both DNA laddering analysis and Tdt-mediated dUTP nick end labeling (TUNEL) assay. Western blot analysis and real-time reverse transcription polymerase chain reaction technologies were applied to assess the expression level of c-Myc, Bax, and Bcl-2 in HT29 cells. KEY FINDINGS Treatment of HT29 cells with celastrol resulted in a growth inhibition effect, and the IC(50) value was 56μM. Celastrol induced HT29 cells apoptosis, and increased the nuclear translocation of beta-catenin. Apoptosis induction effects of celastrol were significantly attenuated by beta-catenin siRNA transfection. Beta-catenin siRNA markedly increased mRNA and protein levels of c-Myc compared with control siRNA. Beta-catenin siRNA significantly inhibited the expression of Bax and Bcl-2 in celastrol-treated HT29 cells. SIGNIFICANCE Beta-catenin mediates the apoptosis induction effects of celastrol in HT29 cells.
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Affiliation(s)
- Wenzong Lu
- Department of Biomedical Engineering, Xi'an Technological University, Xi'an Shaanxi Province, People's Republic of China.
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22
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Pashai N, Hao H, All A, Gupta S, Chaerkady R, De Los Angeles A, Gearhart JD, Kerr CL. Genome-wide profiling of pluripotent cells reveals a unique molecular signature of human embryonic germ cells. PLoS One 2012; 7:e39088. [PMID: 22737227 PMCID: PMC3380858 DOI: 10.1371/journal.pone.0039088] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2012] [Accepted: 05/18/2012] [Indexed: 11/18/2022] Open
Abstract
Human embryonic germ cells (EGCs) provide a powerful model for identifying molecules involved in the pluripotent state when compared to their progenitors, primordial germ cells (PGCs), and other pluripotent stem cells. Microarray and Principal Component Analysis (PCA) reveals for the first time that human EGCs possess a transcription profile distinct from PGCs and other pluripotent stem cells. Validation with qRT-PCR confirms that human EGCs and PGCs express many pluripotency-associated genes but with quantifiable differences compared to pluripotent embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and embryonal carcinoma cells (ECCs). Analyses also identified a number of target genes that may be potentially associated with their unique pluripotent states. These include IPO7, MED7, RBM26, HSPD1, and KRAS which were upregulated in EGCs along with other pluripotent stem cells when compared to PGCs. Other potential target genes were also found which may contribute toward a primed ESC-like state. These genes were exclusively up-regulated in ESCs, IPSCs and ECCs including PARP1, CCNE1, CDK6, AURKA, MAD2L1, CCNG1, and CCNB1 which are involved in cell cycle regulation, cellular metabolism and DNA repair and replication. Gene classification analysis also confirmed that the distinguishing feature of EGCs compared to ESCs, ECCs, and IPSCs lies primarily in their genetic contribution to cellular metabolism, cell cycle, and cell adhesion. In contrast, several genes were found upregulated in PGCs which may help distinguish their unipotent state including HBA1, DMRT1, SPANXA1, and EHD2. Together, these findings provide the first glimpse into a unique genomic signature of human germ cells and pluripotent stem cells and provide genes potentially involved in defining different states of germ-line pluripotency.
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Affiliation(s)
- Nikta Pashai
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
| | - Haiping Hao
- Deep Sequencing and Microarray Core, High Throughput Biology Center, Johns Hopkins University, Baltimore, Maryland, United States of America
| | - Angelo All
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
- Department of Neurology, Johns Hopkins University, Baltimore, Maryland, United States of America
| | - Siddharth Gupta
- Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
| | - Raghothama Chaerkady
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
| | - Alejandro De Los Angeles
- Stem Cell Transplantation Program, Division of Pediatric Hematology Oncology, Children’s Hospital Boston, Massachusetts, United States of America
- Department of Biological Chemistry and Molecular Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America
- Harvard Stem Cell Institute, Cambridge, Massachusetts, United States of America
| | - John D. Gearhart
- Department of Cell and Developmental Biology, Institute of Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
- Department of Animal Biology, Institute of Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - Candace L. Kerr
- Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America
- Department of Gynecology and Obstetrics, Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America
- * E-mail:
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23
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Nomura A, Sugizaki K, Yanagisawa H, Okamoto A. Discrimination between 5-hydroxymethylcytosine and 5-methylcytosine by a chemically designed peptide. Chem Commun (Camb) 2011; 47:8277-9. [PMID: 21695309 DOI: 10.1039/c1cc12131f] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
An artificial phosphopeptide recognized the difference between methylated and hydroxymethylated cytosines in DNA. The Sp1 zinc finger peptide substituted by phosphotyrosine effectively discriminated between 5-methylcytosine, 5-hydroxymethylcytosine ((hm)C) and unmethylated cytosine. The DNA recognition properties of the peptide differ from those of other chemicals that detect (hm)C.
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Affiliation(s)
- Akiko Nomura
- Advanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japan
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24
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Kothinti R, Tabatabai NM, Petering DH. Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA. J Inorg Biochem 2011; 105:569-76. [PMID: 21396899 PMCID: PMC3073834 DOI: 10.1016/j.jinorgbio.2010.08.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2010] [Revised: 08/18/2010] [Accepted: 08/19/2010] [Indexed: 10/19/2022]
Abstract
The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn(2+) and readily competes with zinc finger peptides for Zn(2+) resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn(3)-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn(3)-Sp1 under the conditions of the assay that permit some Zn(3)-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn(3)-Sp1 by GC1, EDTA readily reacted with Zn(3)-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn(3)-Sp1 failed to compete for zinc bound in the Zn(3)-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn(3)-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.
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Affiliation(s)
- Rajendra Kothinti
- Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201-0413, USA
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25
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Quintal SM, dePaula QA, Farrell NP. Zinc finger proteins as templates for metal ion exchange and ligand reactivity. Chemical and biological consequences. Metallomics 2011; 3:121-39. [PMID: 21253649 DOI: 10.1039/c0mt00070a] [Citation(s) in RCA: 84] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Zinc finger reactions with inorganic ions and coordination compounds are as diverse as the zinc fingers themselves. Use of metal ions such as Co(2+) and Cd(2+) has given structural, thermodynamic and kinetic information on zinc fingers and zinc-finger-DNA/RNA interactions. It is a general truism that alteration of the coordination sphere in the finger environment will disrupt the recognition with DNA/RNA and this has implications for mechanism of toxicity and carcinogenesis of metal ions. Structural zinc fingers are susceptible to electrophilic attack and the recognition that the coordination sphere of inorganic compounds may be modulated for control of electrophilic attack on zinc fingers raises the possibility of systematic studies of zinc fingers as drug targets using inorganic chemistry. Some inorganic compounds such as those of As(III) and Au(I) may exert their biological effects through inactivation of zinc fingers and novel approaches to specifically attack the zinc-bound ligands using Co(III)-Schiff bases and Platinum(II)-Nucleobase compounds have been proposed. The genomic importance of zinc fingers suggests that the "coordination chemistry" of zinc fingers themselves is ripe for exploration to design new targets for medicinal inorganic chemistry.
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Affiliation(s)
- Susana M Quintal
- Department of Chemistry, Virginia Commonwealth University, 1001 W. Main St., Richmond, VA 23284-2006, USA
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26
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Cyclic phosphatidic acid decreases proliferation and survival of colon cancer cells by inhibiting peroxisome proliferator-activated receptor γ. Prostaglandins Other Lipid Mediat 2010; 93:126-33. [PMID: 20932931 DOI: 10.1016/j.prostaglandins.2010.09.002] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2010] [Revised: 09/16/2010] [Accepted: 09/25/2010] [Indexed: 12/25/2022]
Abstract
Cyclic phosphatidic acid (cPA), a structural analog of lysophosphatidic acid (LPA), is one of the simplest phospholipids found in every cell type. cPA is a specific, high-affinity antagonist of peroxisome proliferator-activated receptor gamma (PPARγ); however, the molecular mechanism by which cPA inhibits cellular proliferation remains to be clarified. In this study, we found that inhibition of PPARγ prevents proliferation of human colon cancer HT-29 cells. cPA suppressed cell growth, and this effect was reversed by the addition of a PPARγ agonist. These results indicate that the physiological effects of cPA are partly due to PPARγ inhibition. Our results identify PPARγ as a molecular mediator of cPA activity in HT-29 cells, and suggest that cPA and the PPARγ pathway might be therapeutic targets in the treatment of colon cancer.
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27
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Kim E, Jang S, Pak Y. All-atom ab initio native structure prediction of a mixed fold (1FME): a comparison of structural and folding characteristics of various beta beta alpha miniproteins. J Chem Phys 2010; 131:195102. [PMID: 19929079 DOI: 10.1063/1.3266510] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
We performed an all-atom ab initio native structure prediction of 1FME, which is one of the computationally challenging mixed fold beta beta alpha miniproteins, by combining a novel conformational search algorithm (multiplexed Q-replica exchange molecular dynamics scheme) with a well-balanced all-atom force field employing a generalized Born implicit solvation model (param99MOD5/GBSA). The nativelike structure of 1FME was identified from the lowest free energy minimum state and in excellent agreement with the NMR structure. Based on the interpretation of the free energy landscape, the structural properties as well as the folding behaviors of 1FME were compared with other beta beta alpha miniproteins (1FSD, 1PSV, and BBA5) that we have previously studied with the same force field. Our simulation showed that the 28-residue beta beta alpha miniproteins (1FME, 1FSD, and 1PSV) share a common feature of the free energy topography and exhibit the three local minimum states on each computed free energy map, but the 23-residue miniprotein (BBA5) follows a downhill folding with a single minimum state. Also, the structure and stability changes resulting from the two point mutation (Gln1-->Glu1 and Ile7-->Tyr7) of 1FSD were investigated in details for direct comparison with the experiment. The comparison shows that upon mutation, the experimentally observed turn type switch from an irregular turn (1FSD) to type I(') turn (1FME) was well reproduced with the present simulation.
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Affiliation(s)
- Eunae Kim
- Department of Chemistry, Pusan National University, Busan 609-735, Korea
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28
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Maurmann L, Bose RN. Unwinding of zinc finger domain of DNA polymerase I by cis-diamminedichloroplatinum(ii). Dalton Trans 2010; 39:7968-79. [DOI: 10.1039/c0dt00274g] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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29
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Prosperi JR, Becher KR, Willson TA, Collins MH, Witte DP, Goss KH. The APC tumor suppressor is required for epithelial integrity in the mouse mammary gland. J Cell Physiol 2009; 220:319-31. [PMID: 19326388 DOI: 10.1002/jcp.21766] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Inactivation of the adenomatous polyposis coli (APC) tumor suppressor has been associated with mammary tumorigenesis in mouse models and through epidemiological studies of human breast cancers, but the normal role for APC in mammary development has not been thoroughly characterized. We report here that Apc(Min/+) mice containing one functional allele of Apc have severely disrupted lobuloalveolar development during pregnancy and lactation, time points at which Apc gene expression is at its highest levels in normal mice. This phenotype was accompanied by altered proliferation during pregnancy and involution, increased apoptosis throughout lactation, the formation of preneoplastic lesions and changes in specific genes associated with each of these processes. Neither modifications in beta-catenin localization, nor the expression of beta-catenin transcriptional target genes, were observed in Apc(Min/+) mammary tissues; however, tissues from lactating Apc(Min/+) mice had a significantly altered epithelial architecture, including disrupted localization of junctional proteins and polarization. Consistent with these findings, APC knockdown in non-transformed mouse mammary epithelial cells in vitro resulted in altered monolayer formation and proliferation without changes in beta-catenin-mediated transcription. These results suggest that APC expression is tightly regulated during mammary gland development and is required for normal mammary homeostasis and tumor suppression primarily through maintaining epithelial integrity.
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Affiliation(s)
- Jenifer R Prosperi
- Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, USA
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30
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Connors SK, Balusu R, Kundu CN, Jaiswal AS, Gairola CG, Narayan S. C/EBPbeta-mediated transcriptional regulation of bcl-xl gene expression in human breast epithelial cells in response to cigarette smoke condensate. Oncogene 2009; 28:921-32. [PMID: 19043455 PMCID: PMC2642529 DOI: 10.1038/onc.2008.429] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2008] [Revised: 09/18/2008] [Accepted: 10/19/2008] [Indexed: 02/03/2023]
Abstract
In earlier studies, we have shown that cigarette smoke condensate (CSC), a surrogate for cigarette smoke, is capable of transforming the spontaneously immortalized human breast epithelial cell line, MCF10A. These transformed cells displayed upregulation of the anti-apoptotic gene, bcl-xl. Upregulation of this gene may impede the apoptotic pathway and allow the accumulation of DNA damage that can lead to cell transformation and carcinogenesis. In the present study, we have determined the mechanism of CSC-mediated transcriptional upregulation of bcl-xl gene expression in MCF10A cells. We cloned the human bcl-xl promoter (pBcl-xLP) and identified putative transcription factor binding sites. Sequential deletion constructs that removed the putative cis-elements were constructed and transfected into MCF10A cells to determine the CSC-responsive cis-element(s) on the pBcl-xLP. Gel-shift, super-shift and chromatin immunoprecipitation analysis confirmed that CCAAT/enhancer-binding protein (C/EBPbeta) specifically bound to a C/EBP-binding site on the pBcl-xLP in vitro and in vivo. Additionally, overexpression of C/EBPbeta-LAP2 stimulated pBcl-xLP activity and Bcl-xL protein levels, which mimicked the conditions of CSC treatment. Our results indicate that C/EBPbeta regulates bcl-xl gene expression in MCF10A cells in response to CSC treatment; therefore, making it a potential target for chemotherapeutic intervention of cigarette smoke-induced breast carcinogenesis.
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Affiliation(s)
- Shahnjayla K. Connors
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610
| | - Ramesh Balusu
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610
| | - Chanakya N. Kundu
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610
| | - Aruna S. Jaiswal
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610
| | - C. Gary Gairola
- Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536
| | - Satya Narayan
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610
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Jaiswal AS, Narayan S. A novel function of adenomatous polyposis coli (APC) in regulating DNA repair. Cancer Lett 2008; 271:272-80. [PMID: 18662849 PMCID: PMC2585005 DOI: 10.1016/j.canlet.2008.06.024] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2008] [Revised: 04/07/2008] [Accepted: 06/11/2008] [Indexed: 11/22/2022]
Abstract
Prevailing literature suggests diversified cellular functions for the adenomatous polyposis coli (APC) gene. Among them a recently discovered unique role of APC is in DNA repair. The APC gene can modulate the base excision repair (BER) pathway through an interaction with DNA polymerase beta (Pol-beta) and flap endonuclease 1 (Fen-1). Taken together with the transcriptional activation of APC gene by alkylating agents and modulation of BER activity, APC may play an important role in carcinogenesis and chemotherapy by determining whether cells with DNA damage survive or undergo apoptosis. In this review, we summarize the evidence supporting this novel concept and suggest that these results will have implications for the development of more effective strategies for chemoprevention, prognosis and chemotherapy of certain types of tumors.
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Affiliation(s)
- Aruna S. Jaiswal
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, College of Medicine, University of Florida, Gainesville, FL 32610
| | - Satya Narayan
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, College of Medicine, University of Florida, Gainesville, FL 32610
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Kundu CN, Balusu R, Jaiswal AS, Gairola CG, Narayan S. Cigarette smoke condensate-induced level of adenomatous polyposis coli blocks long-patch base excision repair in breast epithelial cells. Oncogene 2007; 26:1428-38. [PMID: 16924228 DOI: 10.1038/sj.onc.1209925] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2006] [Revised: 07/11/2006] [Accepted: 07/11/2006] [Indexed: 11/09/2022]
Abstract
Our previous studies have shown that treatment with cigarette smoke condensate (CSC) transforms normal breast epithelial cell line, MCF-10A. In the present study, the mechanism of CSC-induced transformation of breast epithelial cells was examined. We first determined whether benzo[a]pyrene (B[a]P)- and CSC-induced levels of APC are capable of inhibiting long-patch base excision repair (LP-BER) since our earlier studies had shown that an interaction of APC with DNA polymerase beta (pol-beta) blocks strand-displacement synthesis. With the use of a novel in vivo LP-BER assay, it was demonstrated that increased and decreased APC levels in different breast cancer cell lines were associated with a decrease or increase in LP-BER activity, respectively. The effect of APC on LP-BER in malignant and pre-malignant breast epithelial cell lines was produced by either overexpression or knockdown of APC. Furthermore, it was shown that the decreased LP-BER in B[a]P- or CSC-treated pre-malignant breast epithelial cells is associated with an increased level of APC and decreased cell growth. Our results suggest that the decreased growth allows cells to repair the damaged DNA before mitosis, and failure to repair damaged DNA has the potential to transform pre-malignant breast epithelial cells.
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Affiliation(s)
- C N Kundu
- Department of Anatomy and Cell Biology, UF Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA
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Jaiswal AS, Balusu R, Armas ML, Kundu CN, Narayan S. Mechanism of adenomatous polyposis coli (APC)-mediated blockage of long-patch base excision repair. Biochemistry 2006; 45:15903-14. [PMID: 17176113 PMCID: PMC2528549 DOI: 10.1021/bi0607958] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Recently, we found an interaction between adenomatous polyposis coli (APC) and DNA polymerase beta (pol-beta) and showed that APC blocks strand-displacement synthesis of long-patch base excision repair (LP-BER) (Narayan, S., Jaiswal, A. S., and Balusu, R. (2005) J. Biol. Chem. 280, 6942-6949); however, the mechanism is not clear. Using an in vivo LP-BER assay system, we now show that the LP-BER is higher in APC-/- cells than in APC+/+ cells. In addition to pol-beta, the pull-down experiments showed that the full-length APC also interacted with flap endonuclease 1 (Fen-1). To further characterize the interaction of APC with pol-beta and Fen-1, we performed a domain-mapping of APC and found that both pol-beta and Fen-1 interact with a 138-amino acids peptide from the APC at the DRI-domain. Our functional assays showed that APC blocks pol-beta-mediated 1-nucleotide (1-nt) as well as strand-displacement synthesis of reduced abasic, nicked-, or 1-nt gapped-DNA substrates. Further studies demonstrated that APC blocks 5'-flap endonuclease as well as the 5'-3' exonuclease activity of Fen-1 resulting in the blockage of LP-BER. From these results, we concluded that APC can have three different effects on the LP-BER pathway. First, APC can block pol-beta-mediated 1-nt incorporation and strand-displacement synthesis. Second, APC can block LP-BER by blocking the coordinated formation and removal of the strand-displaced flap. Third, APC can block LP-BER by blocking hit-and-run synthesis. These studies will have important implications for APC in DNA damage-induced carcinogenesis and chemoprevention.
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Affiliation(s)
- Aruna S. Jaiswal
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA
| | - Ramesh Balusu
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA
| | - Melissa L. Armas
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA
| | - Chanakya N. Kundu
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA
| | - Satya Narayan
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA
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Jaiswal AS, Balusu R, Narayan S. 7,12-Dimethylbenzanthracene-dependent transcriptional regulation of adenomatous polyposis coli (APC) gene expression in normal breast epithelial cells is mediated by GC-box binding protein Sp3. Carcinogenesis 2005; 27:252-61. [PMID: 16150893 DOI: 10.1093/carcin/bgi225] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
In the present investigation, we have examined the transcriptional regulation of adenomatous polyposis coli (APC) gene expression in the spontaneously immortalized human normal breast epithelial cell line, MCF10A, in response to carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The APC mRNA levels and the APC gene's promoter (pAPCP) activity were increased in MCF10A cells after treatment with DMBA. A sequential deletion analysis and site-directed mutagenesis of the pAPCP promoter revealed that the DMBA response is mediated through a GC-box element. Also, the GC-box binding agent mithramycin A, which prevents binding of proteins to the GC-box region, abolished DMBA-mediated increase of the pAPCP promoter activity. The specificity of the proteins binding to the GC-box region was characterized by gel-shift analysis. An increased binding of the GC-box binding proteins was observed in the gel-shift analysis with nuclear extracts from DMBA-treated MCF10A cells, which corresponded to the increased levels of Sp1 and Sp3 proteins. However, a super-shift of the DNA-protein complexes was observed with only anti-Sp3 antibody. Based on the chromatin-immunoprecipitation assay results, the Sp3 appeared to be a genuine protein binding to the GC-box site of the pAPCP promoter. In RNA interference experiments, in which the Sp3 expression was knocked down, the DMBA response on the pAPCP promoter activity was reduced, suggesting that the binding of Sp3 to the GC-box site is critical for DMBA-induced pAPCP promoter activity. From these results we conclude that the increased pAPCP promoter activity in the MCF10A cell line in response to DMBA treatment is mediated by Sp3.
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Affiliation(s)
- Aruna S Jaiswal
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA
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Jaiswal AS, Narayan S. Zinc stabilizes adenomatous polyposis coli (APC) protein levels and induces cell cycle arrest in colon cancer cells. J Cell Biochem 2005; 93:345-57. [PMID: 15368361 DOI: 10.1002/jcb.20156] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
In the present study, we investigated the mechanisms by which zinc causes growth arrest in colon cancer cells. The results suggest that zinc treatment stabilizes the levels of the wild-type adenomatous polyposis coli (APC) protein at the post-translational level since the APC mRNA levels and the promoter activity of the APC gene were decreased in HCT-116 cells (which express the wild-type APC gene) after treatment with ZnCl2. Increased levels of wild-type but not truncated APC proteins were required for the ZnCl2-mediated G2/M phase arrest in different colon cancer cell lines. We further tested whether serum-stimulation, which induces cell cycle arrest in the S phase, can relieve ZnCl2-induced G2/M phase arrest of HCT-116 cells. Results showed that in the HCT-116 cells pretreated with ZnCl2, the serum-stimulation neither changed the distribution of G2/M phase arrested cells nor the increased levels of APC protein. The G2/M phase arrest correlated with retarded growth of HCT-116 cells. To further establish that wild-type APC protein plays a role in ZnCl2-induced G2/M arrest, we treated SW480 colon cancer cells that express truncated APC protein. We found that ZnCl2 treatment did not induce G2/M phase arrest in SW480 cells; however, the cell growth was retarded due to the loss of E-cadherin and alpha-tubulin levels. These results suggest that ZnCl2 inhibits the proliferation of colon cancer cells (which carry the wild-type APC gene) through stabilization of the APC protein and cell cycle arrest in the G2/M phase. On the other hand, ZnCl2 inhibits the proliferation of colon cancer cells (which carry the mutant APC gene) by disrupting cellular attachment and microtubule stability.
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Affiliation(s)
- Aruna S Jaiswal
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, College of Medicine, University of Florida, Gainesville, Florida 32610, USA
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Narayan S, Jaiswal AS, Balusu R. Tumor suppressor APC blocks DNA polymerase beta-dependent strand displacement synthesis during long patch but not short patch base excision repair and increases sensitivity to methylmethane sulfonate. J Biol Chem 2004; 280:6942-9. [PMID: 15548520 DOI: 10.1074/jbc.m409200200] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
In the present investigation, we report a previously unsuspected function of the tumor suppressor protein, APC (adenomatous polyposis coli), in the regulation of base excision repair (BER). We identified a proliferating cell nuclear antigen-interacting protein-like box sequence in APC that binds DNA polymerase beta and blocks DNA polymerase beta-mediated strand-displacement synthesis in long patch BER without affecting short patch BER. We further showed that the colon cancer cell line expressing the wild-type APC gene was more sensitive to a DNA-methylating agent due to decreased DNA repair by long patch BER than the cell line expressing the mutant APC gene lacking the proliferating cell nuclear antigen-interacting protein-like box. Experiments based on RNA interference showed that the wild-type APC gene expression is required for DNA methylation-induced sensitivity of colon cancer cells. Thus, APC may play a critical role in determining utilization of long versus short patch BER pathways and affect the susceptibility of colon cancer cells to carcinogenic and chemotherapeutic agents.
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Affiliation(s)
- Satya Narayan
- Department of Anatomy and Cell Biology and Shands Cancer Center, University of Florida, Gainesville, Florida 32610, USA.
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Jaiswal AS, Narayan S. Reduced levels of the adenomatous polyposis coli (APC) protein are associated with ceramide-induced apoptosis of colon cancer cells. J Cancer Res Clin Oncol 2004; 130:695-703. [PMID: 15340841 DOI: 10.1007/s00432-004-0591-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2003] [Accepted: 05/19/2004] [Indexed: 10/26/2022]
Abstract
PURPOSE Mutations of the adenomatous polyposis coli (APC) and p53 genes are commonly found in colorectal cancers. We therefore analyzed the relative roles of APC and p53 in the induction of apoptosis of colon cancer cells by comparing the effects of the natural chemopreventive agent, C(2)-ceramide, on different human colon cancer cell lines with and without wild-type p53 and APC genes. METHODS We studied the effect of C(2)-ceramide and C(2)-dihydroceramide on proliferation and/or apoptosis of colon cancer cell lines in vitro and determined the role of p53 and APC proteins in these processes. The protein and mRNA levels in colon cancer cell lines with and without treatments were determined by Western and Northern blot analysis, respectively. The cell cycle and apoptosis profiles were determined by FACS analysis and PARP-1 cleavage. RESULTS Our findings indicate that C(2)-ceramide can induce apoptosis independently of the p53/p21(Waf-1/Cip-1) pathway. In addition, the C(2)-ceramide induction of apoptosis showed a correlation with a reduction in the levels of the APC protein and mRNA. Moreover, the C(2)-ceramide-induced apoptosis was blocked by pre-treatment with ZnCl(2), which stabilizes APC protein levels. CONCLUSIONS These results suggest that C(2)-ceramide treatment reduces the levels of APC protein and that the reduction in the levels of this protein plays a key role in the ability of C(2)-ceramide to induce apoptosis of colon cancer cells.
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Affiliation(s)
- Aruna S Jaiswal
- Department of Anatomy and Cell Biology, Academic Research Building, College of Medicine, University of Florida, Gainesville, 32610, USA
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Moxley RA, Jarrett HW, Mitra S. Methods for transcription factor separation. J Chromatogr B Analyt Technol Biomed Life Sci 2004; 797:269-88. [PMID: 14630155 DOI: 10.1016/s1570-0232(03)00609-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Recent advances in the separation of transcription factors (TFs) are reviewed in this article. An overview of the transcription factor families and their structure is discussed and a computer analysis of their sequences reveals that while they do not differ from other proteins in molecular mass or isoelectric pH, they do differ from other proteins in the abundance of certain amino acids. The chromatographic and electrophoretic methods which have been successfully used for purification and analysis are discussed and recent advances in stationary and mobile phase composition is discussed.
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Affiliation(s)
- Robert A Moxley
- Department of Biochemistry, 858 Madison Avenue, University of Tennessee Health Science Center, Memphis, TN 38163, USA
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Narayan S, Jaiswal AS, Kang D, Srivastava P, Das GM, Gairola CG. Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro. Oncogene 2004; 23:5880-9. [PMID: 15208684 DOI: 10.1038/sj.onc.1207792] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2004] [Revised: 04/06/2004] [Accepted: 04/06/2004] [Indexed: 12/21/2022]
Abstract
In the present study, we showed that a single-dose treatment of normal breast epithelial cell line, MCF10A, for 72 h with cigarette smoke condensate (CSC) resulted in a transformed phenotype. The anchorage-dependent growth of these cells was decreased due to increased cell cycle arrest in S-G2/M phase; however, the surviving cells developed resistance due to an increased Bcl-xL to Bax ratio. Levels of PCNA and gadd45 proteins--involved in DNA repair in response to genomic damage--were increased, suggesting that the cells were responding to CSC-induced genomic damage. The transformation of MCF10A cells was determined by their colony-forming efficiency in soft-agar in an anchorage-independent manner. CSC-treated MCF10A cells efficiently formed colonies in soft-agar. We then re-established cell lines from the soft-agar colonies and further examined the persistence of their transforming characteristics. The re-established cell lines, when plated after 17 passages without CSC treatment, still formed colonies in the soft-agar. An increased staining of neuropilin-1 (NRP-1) further showed a transformation characteristic of MCF10A cells treated with CSC. In summary, our results suggest that CSC is capable of transforming the MCF10A cells in vitro, supporting the role of cigarette smoking and increased risk for breast cancer.
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Affiliation(s)
- Satya Narayan
- Department of Anatomy and Cell Biology, UF Shands Cancer Center, Academic Research Building, Room R4-216, PO Box 100232, University of Florida, Gainesville, FL 32610, USA.
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Huang M, Shaw CF, Petering DH. Interprotein metal exchange between transcription factor IIIa and apo-metallothionein. J Inorg Biochem 2004; 98:639-48. [PMID: 15041244 PMCID: PMC3535305 DOI: 10.1016/j.jinorgbio.2004.02.004] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2003] [Revised: 01/14/2004] [Accepted: 02/06/2004] [Indexed: 10/26/2022]
Abstract
Zn(2+) and Cd(2+) ion exchange between transcription factor IIIA (TFIIIA) and apo-metallothionein (MT) were studied using a combination of methods including chromatography, ultrafiltration and UV spectroscopy. Under near stoichiometric conditions, apoMT was able to remove most if not all of the zinc ions from TFIIIA, whether or not the TFIIIA was bound to the 5S DNA internal control region (ICR), and concomitantly inhibit its DNA-binding activity as indicated by an electrophoretic mobility shift assay. The kinetics of the two processes were similar. The rate of the metal exchange reaction increased with the concentrations of both reactants. A second-order rate constant of 30+/-10 M(-1)s(-1) was calculated. Similar observations were made for the reaction between apoMT and Cd-substituted TFIIIA, which proceeded without observable intermediates according to a spectrophotometric analysis. A very slow metal ion exchange occurred between Cd-TFIIIA and Zn-MT, but not between Cd-MT and Zn-TFIIIA. Comparative studies on the reaction of TFIIIA with a small competing ligand, ethylenedinitrilo-tetraacetic acid (EDTA), were also conducted. Although EDTA reacts with free Zn-TFIIIA, under similar conditions it failed to compete for Zn(2+) bound as Zn-TFIIIA-ICR.
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Affiliation(s)
| | | | - David H. Petering
- Corresponding author. Tel.: +414-229-5853; fax: +414-229-5530. (D.H. Petering)
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Jaiswal AS, Multani AS, Pathak S, Narayan S. N-methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA. Mol Cancer 2004; 3:3. [PMID: 14728717 PMCID: PMC320492 DOI: 10.1186/1476-4598-3-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2003] [Accepted: 01/16/2004] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is able to induce senescence in the HCT-116 colon cancer cell line. RESULTS Cells treated with lower concentrations of MNNG (0-25 microM) for 50 h showed a dose-dependent increase in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50-100 microM) showed a senescence-like G0/G1 phase arrest which was confirmed by increased expression of beta-galactosidase, a senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of p53 protein, but the senescence-like G0/G1 phase arrest was dissociated with p53 protein levels, since the p53 protein levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-related proteins such as p21, p27, cyclin B1, Cdc2, c-Myc and max. The expression levels of these proteins were increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals with cellular senescence in MNNG-treated cells. We found that protein and mRNA levels of APC were drastically reduced in cells treated with higher concentrations of MNNG. The loss of APC expression might lead to chromosomal instability as well as microtubular disorganization through its dissociation with tubulin. In fact, the protein level of alpha-tubulin was also drastically decreased in senescence-like arrested cells treated with higher concentrations of MNNG. The levels of telomeric DNA also decreased in cells treated with higher concentrations of MNNG. CONCLUSIONS These results suggest that in response to DNA alkylation damage the senescence-like arrest of HCT-116 cells was associated with decreased levels of APC protein, microtubular organization, and telomeric DNA.
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Affiliation(s)
- Aruna S Jaiswal
- UF Shands Cancer Center and Anatomy and Cell Biology, College of Medicine, Academic Research Building, Room R4-216, PO Box 100232, University of Florida, Gainesville, FL 32610, USA
| | - Asha S Multani
- Department of Molecular Genetics, unit & 011, The University, the University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
| | - Sen Pathak
- Department of Molecular Genetics, unit & 011, The University, the University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
| | - Satya Narayan
- UF Shands Cancer Center and Anatomy and Cell Biology, College of Medicine, Academic Research Building, Room R4-216, PO Box 100232, University of Florida, Gainesville, FL 32610, USA
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Narayan S, Roy D. Role of APC and DNA mismatch repair genes in the development of colorectal cancers. Mol Cancer 2003; 2:41. [PMID: 14672538 PMCID: PMC317355 DOI: 10.1186/1476-4598-2-41] [Citation(s) in RCA: 131] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2003] [Accepted: 12/12/2003] [Indexed: 02/06/2023] Open
Abstract
Colorectal cancer is the third most common cause of cancer-related death in both men and women in the western hemisphere. According to the American Cancer Society, an estimated 105,500 new cases of colon cancer with 57,100 deaths will occur in the U.S. in 2003, accounting for about 10% of cancer deaths. Among the colon cancer patients, hereditary risk contributes approximately 20%. The main inherited colorectal cancers are the familial adenomatous polyposis (FAP) and the hereditary nonpolyposis colorectal cancers (HNPCC). The FAP and HNPCC are caused due to mutations in the adenomatous polyposis coli (APC) and DNA mismatch repair (MMR) genes. The focus of this review is to summarize the functions of APC and MMR gene products in the development of colorectal cancers.
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Affiliation(s)
- Satya Narayan
- Department of Anatomy and Cell Biology and UF Shands Cancer Center, College of Medicine, Academic Research Building, Room R4-216, 1600 SW Archer Road, University of Florida, Gainesville, FL 32610, USA
| | - Deodutta Roy
- Environmental Health Sciences, University of Alabama at Birmingham, 317 Ryals Building, 1665 University Boulevard, Birmingham, AL 35294-0022, USA
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Zhu F, Zhang M. DNA polymerase ζ: new insight into eukaryotic mutagenesis and mammalian embryonic development. World J Gastroenterol 2003; 9:1165-9. [PMID: 12800216 PMCID: PMC4611776 DOI: 10.3748/wjg.v9.i6.1165] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Information about the mechanisms that generate mutations in eukaryotes is likely to be useful for understanding human health concerns, such as genotoxicity and cancer. Eukaryotic mutagenesis is largely the outcome of attacks by endogenous and environmental agents. Except for DNA repair, cell cycle checkpoints and DNA damage avoidance, cells have also evolved DNA damage tolerance mechanism, by which lesion-targeted mutation might occur in the genome during replication by specific DNA polymerases to bypass the lesions (translesion DNA synthesis, TLS), or mutation on undamaged DNA templates (untargeted mutation) might be induced. DNA polymerase ζ (pol ζ), which was found firstly in budding yeast Saccharomyces cerevisiae and consists of catalytic subunit scRev3 and stimulating subunit scRev7, has received more attention in recent years. Pol ζ is a member of DNA polymerase δ subfamily, which belongs to DNA polymerase B family, and exists in almost all eukaryotes. Human homolog of the scRev3 gene is located in chromosome region 6q21, and the mouse equivalent maps to chromosome 10, distal to the c-myb gene and close to the Macs gene. Alternative splicing, upstream out-of frame ATG can be found in yeast scRev3, mouse and human homologs. Furthermore, the sequence from 253-323 immediate upstream of the AUG initiator codon has the potential to form a stem-loop hairpin secondary structure in REV3 mRNA, suggesting that human REV3 protein may be expressed at low levels in human cells under normal growth conditions. The functional domain analysis showed that yeast Rev3-980 tyrosine in conserved region II is at the polymerase active site. Human REV3 amino acid residues 1776-2195 provide a REV7 binding domain, and REV7 amino acid residues 1-211 provide a bind domain for REV1, REV3 and REV7 itself. More interestingly, REV7 interacts with hMAD2 and therefore might function in the cell cycle control by affecting the activation of APC (anaphase promoting complex). Currently it has been known that pol ζ is involved in most spontaneous mutation, lesion-targeted mutation via TLS, chemical carcinogen induced untargeted mutation and somatic hypermutation of antibody genes in mammalian. In TLS pathway, pol ζ acts as a "mismatch extender" with combination of other DNA polymerases, such as pol ι. Unlike in yeast, it was found that pol ζ also functioned in mouse embryonic development more recently. It was hypothesized that the roles of pol ζ in TLS and cell cycle control might contribute to mouse embryonic lethality.
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Affiliation(s)
- Feng Zhu
- Department of Pathophysiology, Zhejiang University School of Medicine, Hangzhou 310031, Zhejiang Province, China
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Zhu F, Jin CX, Song T, Yang J, Guo L, Yu YN. Response of human REV3 gene to gastric cancer inducing carcinogen N-methyl- N’-nitro- N-nitrosoguanidine and its role in mutagenesis. World J Gastroenterol 2003; 9:888-93. [PMID: 12717825 PMCID: PMC4611392 DOI: 10.3748/wjg.v9.i5.888] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis.
METHODS: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3- by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3- and FL-REV3-. The blockage effect of REV3 was measured by combination of reverse transcription-polymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3 protein level.
RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P < 0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P < 0.05). In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein. MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P < 0.05) and 5.8 fold (P < 0.01) respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 × 10-6 in FL cells to 0.14 × 10-6 in transgenic cells as well (P < 0.01).
CONCLUSION: The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.
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Affiliation(s)
- Feng Zhu
- Department of Pathophysiology, Zhejiang University School of Medicine, Hangzhou 310031, China.
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Jaiswal AS, Marlow BP, Gupta N, Narayan S. Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells. Oncogene 2002; 21:8414-27. [PMID: 12466962 DOI: 10.1038/sj.onc.1205947] [Citation(s) in RCA: 268] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2002] [Revised: 07/31/2002] [Accepted: 08/07/2002] [Indexed: 12/12/2022]
Abstract
The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.
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Affiliation(s)
- Aruna S Jaiswal
- Department of Anatomy and Cell Biology, College of Medicine, The University of Florida, Gainesville, Florida, FL 32610, USA
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Jaiswal AS, Bloom LB, Narayan S. Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1. Oncogene 2002; 21:5912-22. [PMID: 12185591 DOI: 10.1038/sj.onc.1205789] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2002] [Revised: 06/06/2002] [Accepted: 06/18/2002] [Indexed: 12/31/2022]
Abstract
Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polbeta) or DNA polymerase beta (polbeta) gene-knockout (MEFpolbetaKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G(2)/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polbeta-dependent pathway and by blocking both FEN1 and DNA ligase activity in polbeta-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polbeta-dependent and -independent LP-BER pathways in MEF cells.
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Affiliation(s)
- Aruna S Jaiswal
- Department of Anatomy and Cell Biology, College of Medicine, The University of Florida, Gainesville 32610, USA
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Ahn EH, Schroeder JJ. Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells. Exp Biol Med (Maywood) 2002; 227:345-53. [PMID: 11976405 DOI: 10.1177/153537020222700507] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.
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Affiliation(s)
- Eun Hyun Ahn
- Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824-1224, USA
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Homma MK, Li D, Krebs EG, Yuasa Y, Homma Y. Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein. Proc Natl Acad Sci U S A 2002; 99:5959-64. [PMID: 11972058 PMCID: PMC122884 DOI: 10.1073/pnas.092143199] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2alpha- and beta-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G(2)/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2.
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Affiliation(s)
- Miwako Kato Homma
- Department of Biomolecular Science, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan.
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Jaiswal AS, Narayan S. SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression. Mutat Res 2002; 500:17-30. [PMID: 11890931 DOI: 10.1016/s0027-5107(01)00296-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.
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Affiliation(s)
- Aruna S Jaiswal
- Department of Anatomy and Cell Biology, UF Shands Cancer Center, College of Medicine, University of Florida, P.O. Box 100232, Gainesville, FL 32610, USA
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Thermodynamic evaluation of complexes of zinc and cadmium that mimetize metallic centers in transcription factors. ACTA ACUST UNITED AC 2002. [DOI: 10.1016/s0166-1280(01)00618-2] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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