DOCSLIB.ORG

A Global Suppressor Motif for P53 Cancer Mutants

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Global Suppressor Motif for P53 Cancer Mutants A global suppressor motif for p53 cancer mutants Timothy E. Baroni*†‡, Ting Wang*†§, Hua Qian*†¶, Lawrence R. Dearthʈ, Lan N. Truongʈ, Jue Zengʈ, Alec E. Denes*, Stephanie W. Chen*, and Rainer K. Brachmannʈ** *Department of Medicine, Division of Oncology, Washington University School of Medicine, 660 South Euclid Avenue, P.O. Box 8069, St. Louis, MO 63110; and ʈDepartment of Medicine, Division of Hematology͞Oncology, University of California, Medical Science I, C250, Zot Code 4075, Irvine, CA 92697 Communicated by Stanley J. Korsmeyer, Dana–Farber Cancer Institute, Boston, MA, February 19, 2004 (received for review December 4, 2003) The transcription factor and tumor suppressor protein p53 is fre- changes account for 30% and 50 amino acid changes for 55% of all quently inactivated in human cancers. In many cases, p53 gene core domain mutants with a single amino acid change (5). Thus, mutations result in high levels of inactive, full-length p53 protein with compounds active on a subset of p53 cancer mutants could benefit one amino acid change in the core domain that recognizes p53 a large number of patients. DNA-binding sites. The ability to endow function to mutated p53 Furthermore, as proof of principle, several compounds, amifos- proteins would dramatically improve cancer therapy, because it tine, ellipticine, CP-31398, and PRIMA-1, have already been would reactivate a central apoptotic pathway. By using genetic identified that endow function to a subset of p53 cancer mutants strategies and p53 assays in yeast and mammalian cells, we identified (11–14). The exact rescue mechanisms of and number of p53 a global suppressor motif involving codons 235, 239, and 240. These mutants rescued by these compounds are currently not known. intragenic suppressor mutations, either alone or in combination, CP-31398 and PRIMA-1 were isolated in large-scale drug screens restored function to 16 of 30 of the most common p53 cancer mutants that may be the key to this therapeutic challenge. However, screens tested. The 235–239–240 suppressor motif establishes that manipu- may also fail to identify compounds with the broadest activity, lation of a small region of the core domain is sufficient to activate a because, for example, such compounds were not represented in the large number of p53 cancer mutants. Understanding the structural screened libraries. We have taken a different approach of first basis of the rescue mechanism will allow the pursuit of small com- establishing the existence of a global rescue mechanism for p53 pounds able to achieve a similar stabilization of p53 cancer mutants. cancer mutants through the studies of intragenic suppressor mu- tations and then exploiting these findings for the design of small he transcription factor p53 exerts its tumor suppressor function, compounds. Intragenic suppressor mutations are very instructive, Tin part, by activating target genes after upstream stress signals, because they pinpoint key regions of the p53 core domain that, after such as DNA damage, have been relayed. Final outcomes of p53 modification, provide increased stability to p53 protein. activation are DNA repair, cell-cycle arrest, and͞or apoptosis (1–3). In a pilot study (15), we demonstrated that genetic strategies The 393-aa-long protein has an amino-terminal transactivation using a p53 yeast assay were feasible and yielded intragenic sup- domain, a core domain of Ϸ200 amino acids (amino acids 96–292) pressor mutations. This approach resulted in the rescue of p53 that recognizes p53 DNA-binding sites (DBSs), and a carboxyl- cancer mutant V143A by suppressor amino acid N268D, G245S by terminal tetramerization domain. Estimates suggest that up to half T123P, N239Y or S240N and R249S by the combination of T123A of all human cancers carry p53 gene mutations, of which many have and H168R. Two key questions, however, remained unanswered. been documented in large international databases (4, 5). The Are there additional suppressor regions within the p53 core do- International Agency for Research on Cancer (IARC) TP53 Mu- main? Are the identified suppressor mutations at codons 123, 168, tation Database contains 18,585 somatic p53 mutations (R8, 239͞240, and 268 important for the rescue of other frequent p53 www.iarc.fr͞p53; ref. 5). A total of 13,262, or 71%, of these cancer mutants? Improved tools allowed us to answer these central mutations result in full-length protein with one amino acid substi- questions, and we report here the identification of a global sup- tution within the p53 core domain. pressor motif involving amino acids 235, 239, and 240 that rescues The important functions of p53 and the high frequency of p53 more than half of the most common p53 cancer mutants tested. mutations have resulted in significant interest in exploiting the p53 pathway for novel cancer therapies (6, 7). One particularly appeal- Materials and Methods ing approach is the use of small compounds for the pharmacological PCR Mutagenesis of the Upstream and Downstream Regions of p53 restoration of p53 function to those cancers that carry full-length Cancer Mutations. PCR products covering the core domain regions p53 protein with one amino acid change in the DNA-binding core upstream or downstream of a cancer mutation were obtained under domain (8). This strategy, if realized, should have several significant mutagenic conditions (16, 17) and were transformed with appro- advantages. The number of patients that may potentially benefit is priately gapped mutant p53 expression plasmids into the p53 yeast quite large. Based on estimates by the IARC of new cancer cases reporter strain RBy377. RBy377 contains two copies of the p53- worldwide for the year 2000 (www-dep.iarc.fr͞globocan͞ dependent reporter gene 1cUAS53::URA and resulted from a globocan.html; ref. 9), estimates for the frequency of p53 mutations mating of RBy41 (18, 19) and RBy171 (MAT ␣ his3⌬200 leu2⌬1 in specific cancers (10) and the IARC TP53 Mutation Database (5), 1cUAS53::URA), selection for diploids on SC-His-Lys plates (lack- 10 million people are diagnosed with cancer every year, and 2.6 ing histidine and lysine) and segregation of pRB16. Yeast trans- million of them have cancers with a core domain missense muta- formants that had repaired the p53 plasmid by homologous recom- tion. In addition, p53 mutations are frequently found in solid tumors, such as lung, colon, head and neck, and pancreas cancers, that are quite resistant to conventional therapies (4, 5). Such Abbreviations: DBS, DNA-binding site; IARC, International Agency for Research on Cancer. compounds should, in theory, only have an effect on cancer cells, †T.E.B., T.W., and H.Q. contributed equally to this work. because the core domains of WT p53 in normal cells are already ‡Present address: Division of Genetic Disorders, Wadsworth Center, 120 New Scotland structurally intact. Furthermore, the approach promises systemic Avenue, Albany, NY 12084. delivery and lack of a host immune response. §Present address: Department of Genetics, Washington University School of Medicine, 660 The small compound strategy, however, poses formidable chal- South Euclid Avenue, P.O. Box 8232, St. Louis, MO 63110. lenges. Such compounds simply may not exist, or, if they do, may be ¶Present address: Department of Surgery, University of Western Ontario, 339 Windermere effective against only a small subset of the nearly 1,000 reported p53 Road, LHSC-UC 9L14, London, ON, Canada N6A 5A5. core domain mutants. However, from a clinical perspective, the **To whom correspondence should be addressed. E-mail: [email protected]. problem may not be quite as complex, because only 8 amino acid © 2004 by The National Academy of Sciences of the USA 4930–4935 ͉ PNAS ͉ April 6, 2004 ͉ vol. 101 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401162101 Downloaded by guest on September 30, 2021 bination were selected on plates lacking histidine (circular plasmid Table 1. Intragenic suppressor mutations obtained with required) and uracil (expression of the URA3 reporter gene re- PCR mutagenesis quired). Single-colony-purified HisϩUraϩ yeast clones were tested ϩ Cancer for plasmid-dependency of the Ura phenotype. Retention of the mutation No. Suppressor mutations cancer mutation on the p53 plasmid was verified by yeast colony PCR, followed by digestion of the PCR product to identify the G245S 1 F113L unique restriction site provided by the cancer mutation codon. 2 L114V ϩ T123P* ϩ V172l ϩ A189V Positive plasmids were then rescued from yeast, were sequenced 3 T123P* ϩ A189V and were transformed again into RBy377 for phenotype confirma- 4 S227P ϩ N239Y* tion (15). Between 1 ϫ 106 to 1 ϫ 107 yeast transformants per region 5 N239Y* ϩ were analyzed (see Supporting Materials and Methods, which is R249S 6 T118M H168R* ϩ ϩ published as supporting information on the PNAS web site, for 7 V122I C124S H168R* ϩ ϩ more details, including the construction and characterization of 8 K139R H168R* N239Y* ϩ yeast and mammalian plasmids). 9 H168R* T231I R273C 10 T123A* ϩ S240R ϩ Oligonucleotide-Mediated Mutagenesis of p53 Codons 225–241. The 11 H178Y S240R ϩ ϩ ϩ saturation mutagenesis of codons 239 and 240 depended on our 12 D281G E285G G325R E343V ϩ ϩ p53 13 E285K E294G E346G ability to create a short gap in mutant plasmids by using ϩ ϩ restriction enzymes PflMI and NsiI. The second NsiIsiteinthe R273H 14 T81S A83V S240R 15 P87R ϩ Q100L ϩ Q104P ϩ Q144L ϩ S240R backbone of pRS413 was therefore destroyed, and all expression ϩ cassettes for p53 mutants were transferred into this new backbone 16 S183T S240R 17 E224G ϩ S240R by using ApaI–SacI. Thirty-eight annealed primer pairs were gen- 18 H233R ϩ S240R erated, each of them representing a different amino acid change at 19 S240R codon 239 or 240. Equal amounts of each primer pair were cloned separately into the same gapped p53 plasmid, and the number of All clones shown are derived from independent PCR-mediated mutational Escherichia coli clones was determined.
Load more

Recommended publications
  • Wild-Type P53 Can Down-Modulate the Activity of Various Promoters
    Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9979-9983, November 1991 Biochemistry Wild-type p53 can down-modulate the activity of various promoters DORON GINSBERG*, FATIMA MECHTAt, MOSHE YANIVt, AND MOSHE OREN*t *Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel; and tDepartment of Biotechnology, Pasteur Institute, 75724 Paris Cedex 15, France Communicated by Robert A. Weinberg, August 16, 1991 ABSTRACT The wild-type (wt) p53 protein is the product out whether it could also modulate c-fos expression. We of a tumor suppressor gene that is a frequent target for report here that wt p53 can inhibit c-fos gene expression. This inactivation in many types of tumors. The nuclear localization effect of wt p53 is rapid, suggesting that it may precede ofthe protein, as well as additional features, suggest that it may growth arrest. In addition, cotransfection experiments indi- be involved in the regulation ofgene expression. To explore this cate that wt p53 can down-modulate the activity of a number possibility, the effects of overproduced wt p53 were investi- of promoters. While the effect of wt p53 appeared to be gated in a number of systems. Induction of growth arrest via relatively nonspecific, it probably did not reflect a general- the antiproliferative effect ofwt p53 greatly impaired the ability ized transcriptional shut-off. Our findings suggest that wt p53 of cells to exhibit an increase in c-fos mRNA upon serum may contribute to growth inhibition by down-modulating, stimulation. Experiments in which cells were cotransfected directly or indirectly, the expression of genes that are re- with p53 expression plasmids together with a reporter gene quired for ongoing cell proliferation.
    [Show full text]
  • Original Article Regulation of Exogenous P53 Combined with Dickkopf-1 on Human Osteosarcoma MG-63 Cells
    Int J Clin Exp Med 2017;10(6):9252-9258 www.ijcem.com /ISSN:1940-5901/IJCEM0052476 Original Article Regulation of exogenous P53 combined with Dickkopf-1 on human osteosarcoma MG-63 cells Fuyu Gao, Benjun Bi, Guangjun Liao Department of Orthopedic Surgery, Yantaishan Hospital, Yantai, Shandong, China Received March 8, 2017; Accepted April 22, 2017; Epub June 15, 2017; Published June 30, 2017 Abstract: Objective: To regulate the growth of human osteosarcoma cell strain MG-63 in vitro through the combined drugs of exogenous P53 gene and Dickkopf-1 and to explore its possible mechanisms in order to provide the refer- ence for clinical treatment of osteosarcoma. Methods: Human osteosarcoma MG-63 cell strain was subcultured in vitro, and exogenous P53 and Dickkopf-1 were used separately or jointly to act on MG-63 cells. The proliferation and apoptosis of MG-63 cells were respectively detected by trypan blue exclusion and flow cytometry; the expres- sion levels of tumor suppressor gene P53 and oncogene c-fos and c-myc were detected by real-time fluorescent quantitative and western blot in mRNA and protein levels. Results: The proliferation test results showed that the combination of exogenous P53 and Dickkopf-1 could significantly inhibit the growth of MG-63 cells, compared with the single drug using (P<0.05), and cell cycle assay found that the combined drugs caused the cell cycle arrest at G0/G1 phase (P<0.05). Flow cytometry assay indicated the apoptosis of MG-63 cells caused by the combined drugs was significantly higher than that of using exogenous P53 or Dickkopf-1 alone, and the results of western blot manifested the combined drugs could down-regulate the expression levels of c-fos and c-myc whichwere related to the proliferation and differentiation of MG-63 cells, and increase the expression of tumor suppressor gene P53.
    [Show full text]
  • The Screening of the Second-Site Suppressor Mutations of The
    Int. J. Cancer: 121, 559–566 (2007) ' 2007 Wiley-Liss, Inc. The screening of the second-site suppressor mutations of the common p53 mutants Kazunori Otsuka, Shunsuke Kato, Yuichi Kakudo, Satsuki Mashiko, Hiroyuki Shibata and Chikashi Ishioka* Department of Clinical Oncology, Institute of Development, Aging and Cancer, and Tohoku University Hospital, Tohoku University, Sendai, Japan Second-site suppressor (SSS) mutations in p53 found by random mutation library covers more than 95% of tumor-derived missense mutagenesis have shown to restore the inactivated function of mutations as well as previously unreported missense mutations. some tumor-derived p53. To screen novel SSS mutations against We have shown the interrelation among the p53 structure, the common mutant p53s, intragenic second-site (SS) mutations were function and tumor-derived mutations. We have also observed that introduced into mutant p53 cDNA in a comprehensive manner by the missense mutation library was useful to isolate a number of using a p53 missense mutation library. The resulting mutant p53s 16,17 with background and SS mutations were assayed for their ability temperature sensitive p53 mutants and super apoptotic p53s. to restore the p53 transactivation function in both yeast and Previous studies have shown that there are second-site (SS) mis- human cell systems. We identified 12 novel SSS mutations includ- sense mutations that recover the inactivated function of tumor- ing H178Y against a common mutation G245S. Surprisingly, the derived mutations, so-called intragenic second-site suppressor G245S phenotype is rescued when coexpressed with p53 bearing (SSS) mutations.18–23 Analysis of the molecular background of the H178Y mutation.
    [Show full text]
  • Carcinogeninduced Hepatic Tumors in KLF6+/ Mice Recapitulate
    HEPATOBILIARY MALIGNANCIES Carcinogen-Induced Hepatic Tumors in KLF61/2 Mice Recapitulate Aggressive Human Hepatocellular Carcinoma Associated with p53 Pathway Deregulation Mirko Tarocchi,1,2 Rebekka Hannivoort,1,3 Yujin Hoshida,4 Ursula E. Lee,1 Diana Vetter,1 Goutham Narla,5,6 Augusto Villanueva,7,8 Moshe Oren,8 Josep M. Llovet,1,7,9 and Scott L. Friedman1 Inactivation of KLF6 is common in hepatocellular carcinoma (HCC) associated with hepa- titis C virus (HCV) infection, thereby abrogating its normal antiproliferative activity in liver cells. The aim of the study was to evaluate the impact of KLF6 depletion on human HCC and experimental hepatocarcinogenesis in vivo. In patients with surgically resected HCC, reduced tumor expression of KLF6 was associated with decreased survival. Consistent with its role as a tumor suppressor, KLF61/2 mice developed significantly more tumors in response to the chemical carcinogen diethyl nitrosamine (DEN) than wild-type animals. Gene expression signatures in both surrounding tissue and tumors of KLF61/2 mice closely recapitulated those associated with aggressive human HCCs. Expression microarray profiling also revealed an increase in Mdm2 mRNA in tumors from KLF61/2 compared with KLF61/1 mice, which was validated by way of quantitative real-time polymerase chain reaction and western blot analysis in both human HCC and DEN-induced murine tumors. Moreover, chromatin immunoprecipitation and cotransfection assays established the P2 intronic promoter of Mdm2 as a bona fide transcriptional target repressed by KLF6. Whereas KLF6 overexpression in HCC cell lines and primary hepatocytes led to reduced MDM2 levels and increased p53 protein and transcriptional activity, reduction in KLF6 by small interfering RNA led to increased MDM2 and reduced p53.
    [Show full text]
  • Antibiotic Resistance by High-Level Intrinsic Suppression of a Frameshift Mutation in an Essential Gene
    Antibiotic resistance by high-level intrinsic suppression of a frameshift mutation in an essential gene Douglas L. Husebya, Gerrit Brandisa, Lisa Praski Alzrigata, and Diarmaid Hughesa,1 aDepartment of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala SE-751 23, Sweden Edited by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved January 4, 2020 (received for review November 5, 2019) A fundamental feature of life is that ribosomes read the genetic (unlikely if the DNA sequence analysis was done properly), that the code in messenger RNA (mRNA) as triplets of nucleotides in a mutants carry frameshift suppressor mutations (15–17), or that the single reading frame. Mutations that shift the reading frame gen- mRNA contains sequence elements that promote a high level of ri- erally cause gene inactivation and in essential genes cause loss of bosomal shifting into the correct reading frame to support cell viability viability. Here we report and characterize a +1-nt frameshift mutation, (10). Interest in understanding these mutations goes beyond Mtb and centrally located in rpoB, an essential gene encoding the beta-subunit concerns more generally the potential for rescue of mutants that ac- of RNA polymerase. Mutant Escherichia coli carrying this mutation are quire a frameshift mutation in any essential gene. We addressed this viable and highly resistant to rifampicin. Genetic and proteomic exper- by isolating a mutant of Escherichia coli carrying a frameshift mutation iments reveal a very high rate (5%) of spontaneous frameshift suppres- in rpoB and experimentally dissecting its genotype and phenotypes. sion occurring on a heptanucleotide sequence downstream of the mutation.
    [Show full text]
  • Teacher Background on P53 Tumor Suppressor Protein
    Cancer Lab p53 – Teacher Background on p53 Tumor Suppressor Protein Note: The Teacher Background Section is meant to provide information for the teacher about the topic and is tied very closely to the PowerPoint slide show. For greater understanding, the teacher may want to play the slide show as he/she reads the background section. For the students, the slide show can be used in its entirety or can be edited as necessary for a given class. What Is p53 and Where Is the Gene Located? While commonly known as p53, the official name of this gene is Tumor Protein p53 and its official symbol is TP53. TheTP53 gene codes for the TP53 (p53) protein which acts as a tumor suppressor and works in response to DNA damage to orchestrate the repair of damaged DNA. If the DNA cannot be repaired, the p53 protein prevents the cell from dividing and signals it to undergo apoptosis (programmed cell death). The name p53 is due to protein’s 53 kilo-Dalton molecular mass. The gene which codes for this protein is located on the short (p) arm of chromosome 17 at position 13.1 (17p13.1). The gene begins at base pair 7,571,719 and ends at base pair 7, 590,862 making it 19,143 base pairs long. (1, 2) What Does the p53 Gene Look Like When Translated Into Protein? The TP53 gene has 11 exons and a very large 10 kb intron between exons 1 and 2. In humans, exon 1 is non-coding and it has been shown that this region could form a stable stem-loop structure which binds tightly to normal p53 but not to mutant p53 proteins.
    [Show full text]
  • Jdp2 Downregulates Trp53 Transcription to Promote Leukaemogenesis in the Context of Trp53 Heterozygosity
    Oncogene (2013) 32, 397 --402 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc SHORT COMMUNICATION Jdp2 downregulates Trp53 transcription to promote leukaemogenesis in the context of Trp53 heterozygosity L van der Weyden1, AG Rust1,4, RE McIntyre1,4, CD Robles-Espinoza1, M del Castillo Velasco-Herrera1, R Strogantsev2, AC Ferguson-Smith2, S McCarthy1, TM Keane1, MJ Arends3 and DJ Adams1 We performed a genetic screen in mice to identify candidate genes that are associated with leukaemogenesis in the context of Trp53 heterozygosity. To do this we generated Trp53 heterozygous mice carrying the T2/Onc transposon and SB11 transposase alleles to allow transposon-mediated insertional mutagenesis to occur. From the resulting leukaemias/lymphomas that developed in these mice, we identified nine loci that are potentially associated with tumour formation in the context of Trp53 heterozygosity, including AB041803 and the Jun dimerization protein 2 (Jdp2). We show that Jdp2 transcriptionally regulates the Trp53 promoter, via an atypical AP-1 site, and that Jdp2 expression negatively regulates Trp53 expression levels. This study is the first to identify a genetic mechanism for tumour formation in the context of Trp53 heterozygosity. Oncogene (2013) 32, 397--402; doi:10.1038/onc.2012.56; published online 27 February 2012 Keywords: p53; Jdp2; transposon; heterozygosity; lymphoma; mice INTRODUCTION targeted by transposon insertion events leading to upregulated Genetic alterations of TP53 are frequent events in tumourigenesis Jdp2 expression and a decrease in Trp53 expression levels. Further and promote genomic instability, impair apoptosis, and contribute we illustrate that Jdp2 regulates the Trp53 promoter via an atypical to aberrant self-renewal.1--4 The spectrum of mutations that occur AP-1 binding site.
    [Show full text]
  • RB1 and P53 at the Crossroad of EMT and Triple Negative Breast Cancer
    PERSPECTIVE PERSPECTIVE Cell Cycle 10:10, 1-8; May 15, 2011; © 2011 Landes Bioscience RB1 and p53 at the crossroad of EMT and triple negative breast cancer Zhe Jiang,1 Robert Jones,1 Jeff C. Liu,1 Tao Deng,1 Tyler Robinson,1 Philip E.D. Chung,1 Sharon Wang,1 Jason I. Herschkowitz,2 Sean E. Egan,3 Charles M. Perou4 and Eldad Zacksenhaus1,* 1Division of Cell and Molecular Biology; Toronto General Research Institute; University Health Network; Toronto, Ontario, Canada; 2Department of Molecular and Cellular Biology; Baylor College of Medicine; Houston, TX USA; 3Program in Developmental and Stem Cell Biology; The Hospital for Sick Children; Department of Molecular Genetics; University of Toronto; Toronto, Ontario, Canada; 4Lineberger Comprehensive Cancer Center; Department of Genetics and Pathology; University of North Carolina at Chapel Hill; Chapel Hill, NC USA riple negative breast cancer (TNBC) NEU-positive and Triple Negative (TN) Tis a heterogeneous disease that tumors, the latter of which do not express includes Basal-like and Claudin-low hormone receptors or HER2.1-7 TNBC tumors. The Claudin-low tumors are affects 15–30% of patients. By IHC it can enriched for features associated with be further divided into Basal-like breast epithelial-to-mesenchymal transition cancer and non-basal tumors, some of (EMT) and possibly for tumor initiating which exhibit features of EMT.8 Basal-like cells. Primary TNBCs respond relatively BCs express the basal cytokeratins (CK) well to conventional chemotherapy; CK5/6, CK14, CK17, and/or epidermal © 2011 Landes Bioscience. Landes ©2011 however, metastatic disease is virtually growth factor receptor (EGFR), whereas incurable.
    [Show full text]
  • Crucial Role of C-Jun Phosphorylation at Ser63/73 Mediated by PHLPP Protein Degradation in the Cheliensisin a Inhibition of Cell Transformation
    Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233 Cancer Prevention Research Article Research Crucial Role of c-Jun Phosphorylation at Ser63/73 Mediated by PHLPP Protein Degradation in the Cheliensisin A Inhibition of Cell Transformation Junlan Zhu1,2, Jingjie Zhang1, Haishan Huang1,2, Jingxia Li1, Yonghui Yu1, Honglei Jin1,2, Yang Li1,2, Xu Deng3, Jimin Gao2, Qinshi Zhao3, and Chuanshu Huang1 Abstract Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the current study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP downregulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A, and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A.
    [Show full text]
  • The Rich World of P53 DNA Binding Targets: the Role of DNA Structure
    International Journal of Molecular Sciences Review The Rich World of p53 DNA Binding Targets: The Role of DNA Structure Václav Brázda * and Miroslav Fojta Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 612 65 Brno, Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-541-517-231 Received: 11 October 2019; Accepted: 8 November 2019; Published: 9 November 2019 Abstract: The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with DNA. p53 works as a transcription factor for a significant number of genes. Most p53 target genes contain so-called p53 response elements in their promoters, consisting of 20 bp long canonical consensus sequences. Compared to other transcription factors, which usually bind to one concrete and clearly defined DNA target, the p53 consensus sequence is not strict, but contains two repeats of a 50RRRCWWGYYY30 sequence; therefore it varies remarkably among target genes. Moreover, p53 binds also to DNA fragments that at least partially and often completely lack this consensus sequence. p53 also binds with high affinity to a variety of non-B DNA structures including Holliday junctions, cruciform structures, quadruplex DNA, triplex DNA, DNA loops, bulged DNA, and hemicatenane DNA. In this review, we summarize information of the interactions of p53 with various DNA targets and discuss the functional consequences of the rich world of p53 DNA binding targets for its complex regulatory functions. Keywords: p53; protein-DNA interactions; consensus sequence; cruciform; local DNA structures 1. Introduction p53 is the most often mutated tumor suppressor in humans and is studied intensively from different points of view due to its crucial role in malignant transformation [1–3].
    [Show full text]
  • Androgen-Modulated P21 and P53 Gene Expression in Human Non-Transformed Epithelial Prostatic Cells in Primary Cultures
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 30: 967-973, 2012 Androgen-modulated p21 and p53 gene expression in human non-transformed epithelial prostatic cells in primary cultures A. POZZOBON1, L. SCHNEIDER2 and I.S. BRUM2 1Center for Health Sciences, University Center Univates, Lajeado, State of Rio Grande do Sul; 2Laboratory of Tumor and Endocrine Biology, Federal University of Rio Grande do Sul, Porto Alegre, State of Rio Grande do Sul, Brazil Received May 4, 2012; Accepted July 13, 2012 DOI: 10.3892/ijmm.2012.1082 Abstract. The prostate gland is under androgen control. The prostate carcinoma (PCa), CWR22R3, maintains a high level aim of the present study was to evaluate the expression of of AR expression. These cells were infected with retroviruses two genes that are regulators of the cell cycle, the p53 and encoding AR-specific short hairpin RNA (shRNA), and AR p21 genes, in human non-transformed epithelial prostatic cells downregulation was demonstrated by anti-AR immunoblot- (HNTEPs) treated with different concentrations of hormones. ting; also, the cells expressing AR shRNA showed reduced Samples of prostate tissue were obtained from 10 patients cell density (6). Finally, blocking AR activity is a therapeutic between 60 and 77 years of age. HNTEP cells were grown approach for benign prostate hyperplasia (BPH) and PCa (7,8). in basal medium and treated with dihydrotestosterone (DHT) While the stimulatory effect of androgens on in vivo in different conditions for 4 h. A low concentration of DHT growth and development is well recognized in several animal resulted in a significant increase in cell growth; this effect was models and in human prostate, the in vitro characterization of eradicated by addition of the antiandrogen hydroxyflutamide.
    [Show full text]
  • Regulation of the P53 Transcriptional Response by Structurally Diverse Core Promoters
    Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of the p53 transcriptional response by structurally diverse core promoters Jose´ M. Morachis, Christopher M. Murawsky, and Beverly M. Emerson1 Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, we examined the role of core promoter structures in this process. We find that the p21 core promoter directs rapid, TATA box- dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. We define a downstream element within the Fas/APO1 core promoter that is essential for its activation, and identify nuclear transcription factor Y (NF-Y) as its binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress.
    [Show full text]