scholarly journals Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding.

1992 ◽  
Vol 12 (12) ◽  
pp. 5581-5592 ◽  
Author(s):  
E Shaulian ◽  
A Zauberman ◽  
D Ginsberg ◽  
M Oren
Keyword(s):  
Dna Binding ◽  
Point Mutation ◽  
P53 Gene ◽  
Dominant Negative ◽  
Mutant P53 ◽  
Wild Type ◽  
Transforming Activity ◽  
Wild Type P53 ◽  
Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding

1992 ◽  
Vol 12 (12) ◽  
pp. 5581-5592 ◽  
Author(s):  
E Shaulian ◽  
A Zauberman ◽  
D Ginsberg ◽  
M Oren
Keyword(s):  
Dna Binding ◽  
Point Mutation ◽  
P53 Gene ◽  
Dominant Negative ◽  
Mutant P53 ◽  
Wild Type ◽  
Transforming Activity ◽  
Wild Type P53 ◽  
Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

Wild Type p53 Gene Sensitizes Rat C6 Glioma Cells to HSV-TK/ACV Treatment In Vitro and In Vivo

2010 ◽  
Vol 16 (4) ◽  
pp. 509-514 ◽  
Author(s):  
Qiang Huang ◽  
Zhibo Xia ◽  
Yongping You ◽  
Peiyu Pu
Keyword(s):  
Glioma Cells ◽  
P53 Gene ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
Wild Type ◽  
Wild Type P53 ◽  
Rat C6 Glioma

scholarly journals Expression of Wild-Type p53 by Curcumin, Alpinetin and Flavokawain B in Colorectal Cancer cells Expressing R273H Mutant p53

2020 ◽  
Vol 27 (2) ◽  
pp. 88-94
Author(s):  
I. Malami ◽  
A. Muhammad ◽  
I.B. Abubakar ◽  
A.M. Alhassan
Keyword(s):  
Bioactive Compounds ◽  
Mutant P53 ◽  
Wild Type ◽  
P53 Expression ◽  
Gene And Protein Expression ◽  
Colorectal Cancer Cells ◽  
Binding Core ◽  
Wild Type P53 ◽  
Dose Dependent

A mutation in p53 is frequently reported in nearly 50% of all of human cancers arising from DNA-binding core domain of p53. DNA-contact mutant R273H rendered p53 at dysfunctional state due to the substitution of single residue Arg273 for His273. Here, natural bioactive compounds curcumin, alpinetin and flavokawain B were investigated for possible stabilisation of wild-type p53 expression in vitro using HT-29 cells harbouring R273H rendered p53. Accordingly, all the bioactive compounds were able to induce the expression of wild-type p53 both at the levels of gene and protein expression. A dose-dependent induction of p53 was evident at 12.5, 25 and 50 μM concentration. The present study has shown that the bioactive compounds may have restored the wild-type p53 functional activity in tumour cells expressing R273H mutant p53. Keywords: Curcumin, Alpinetin, Flavokawain B, p53, R273H

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Adenovirus mediated p53 tumour suppressor gene therapy for human gastric cancer cells in vitro and in vivo

Gut ◽  
1999 ◽  
Vol 44 (3) ◽  
pp. 366-371 ◽  
Author(s):  
M Ohashi ◽  
F Kanai ◽  
H Ueno ◽  
T Tanaka ◽  
K Tateishi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Gene Therapy ◽  
P53 Gene ◽  
Human Gastric Cancer ◽  
Wild Type ◽  
Gastric Cancer Cells ◽  
Gastric Cancer Cell Lines ◽  
Wild Type P53

BACKGROUND/AIMSGastric cancer is one of the most prevalent forms of cancer in East Asia. Point mutation of the p53 gene has been reported in more than 60% of cases of gastric cancer and can lead to genetic instability and uncontrolled cell proliferation. The purpose of this investigation was to evaluate the potential of p53 gene therapy for gastric cancer.METHODSThe responses of human gastric cancer cell lines, MKN1, MKN7, MKN28, MKN45, and TMK-1, to recombinant adenoviruses encoding wild type p53 (AdCAp53) were analysed in vitro. The efficacy of the AdCAp53 treatment for MKN1 and MKN45 subcutaneous tumours in nude mice was assessed in vivo.RESULTSp53-specific growth inhibition was observed in vitro in two of four gastric cancer cell lines with mutated p53, but not in the wild type p53 cell line. The mechanism of the killing of gastric cancer cells by AdCAp53 was found, by flow cytometric analysis and detection of DNA fragmentation, to be apoptosis. In vivo studies showed that the growth of subcutaneous tumours of p53 mutant MKN1 cells was significantly inhibited by direct injection of AdCAp53, but no significant growth inhibition was detected in the growth of p53 wild type MKN45 tumours.CONCLUSIONSAdenovirus mediated reintroduction of wild type p53 is a potential clinical utility in gene therapy for gastric cancers.

Effects of Gamma Knife surgery on C6 glioma in combination with adenoviral p53 in vitro and in vivo

2006 ◽  
Vol 105 (Supplement) ◽  
pp. 208-213 ◽  
Author(s):  
Desheng Xu ◽  
Qiang Jia ◽  
Yanhe Li ◽  
Chunsheng Kang ◽  
Peiyu Pu
Keyword(s):  
Glioma Cells ◽  
P53 Gene ◽  
Treated Group ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
Survival Duration ◽  
Wild Type ◽  
Wild Type P53

ObjectThe authors sought to study the combined potential of wild-type p53 gene transfer and Gamma Knife surgery (GKS) for the treatment of glioblastomas multiforme. Modification of the radiation response in C6 glioma cells in vitro and in vivo by the wild-type p53 gene was investigated.MethodsStable expression of wild-type p53 in C6 cells was achieved by transduction of the cells with adenoviral p53. Two days later, some cells were treated with GKS. Forty-eight hours after irradiation, the comparative survival rate was assessed by monotetrazolium (MTT) assays. Treated and control C6 glioma cells (4 × 103 per well) were plated into a 96-well plate in octuplicate and tested every 24 hours. Meanwhile, immunohistopathological examination of proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate (TUNEL) assays were performed. The MTT assays indicated the p53, GKS, and combined treated cells proliferated at a significantly lower rate than those of the control group (p < 0.01, Days 2–6) and the positive fraction of PCNA in p53-treated group and GKS-treated group was 70.18 ± 3.61 and 50.71 ± 2.61, respectively, whereas the percentage in the combined group was 30.68 ± 1.49 (p < 0.01).Fifty-six male Sprague–Dawley rats were anesthetized and inoculated with 106 cultured C6 glioma cells into the cerebrum. Forty-eight hours after transduction with adenoviral p53, some rats underwent GKS. A margin dose of 15 Gy was delivered to the 50% isodose line. Two days later, six rats in each group were killed. Their brains were removed and paraffin-embedded section were prepared for immunohistopathological examination and TUNEL assays. The remaining rats were observed for the duration of the survival period. The survival curve indicated that a modest but significant enhancement of survival duration was seen in the p53-treated or GKS alone groups, whereas a more marked and highly significant enhancement of survival duration was achieved when these two treatment modalities were combined. When PCNA expression was downregulated, apoptotic cells become obvious after TUNEL staining.Conclusions The findings of this study suggest that p53-based gene therapy in combination with GKS may be superior to single-modality treatment of C6 glioma.

p53 domains: structure, oligomerization, and transformation

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

HOXA11 Mutation in Amegakaryocytic Thrombocytopenia with Radio-Ulnar Synostosis Syndrome Inhibits Megakaryocytic Differentiation In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1751-1751 ◽  
Author(s):  
Regina D. Horvat-Switzer ◽  
Alexis A. Thompson
Keyword(s):  
Dna Binding ◽  
Point Mutation ◽  
Fold Increase ◽  
Physical Interaction ◽  
Wild Type ◽  
Mobility Shift ◽  
Transfected Cells ◽  
Megakaryocytic Differentiation ◽  
Dnase Treatment

Abstract Homeobox genes encode for regulatory proteins central to bone morphogenesis as well as hematopoietic differentiation and proliferation. Previously we have identified an inherited syndrome of congenital amegakaryocytic thrombocytopenia and radio-ulnar synostosis that is associated with a point mutation in the third helix of HOXA11 homeodomain (HOXA11-ΔH3). We postulated that this mutation results in a truncated protein with impaired DNA binding efficiency. Compared to wild type (HOXA11-WT) in vitro transcription and translation of HOXA11-ΔH3 produced a truncated form of the protein. Electropheretic mobility shift assays (EMSA) confirmed that product from a HoxA11-WT cDNA expression plasmid interacts with the DNA binding consensus sequence for HoxA11 and this interaction is most efficient when its TALE transcription co-factor, Meis1b, is also present. Using the truncated HOXA11-ΔH3 product, the mobility shift was abrogated even in the presence of Meis1b, suggesting the point mutant causes a disruption in the DNA binding capacity of HoxA11. We investigated whether the point mutation affected the physical protein-protein interaction that occurs between HoxA11 and Meis1b. Using GST-pulldown assays we examined these interactions. Meis1b showed a 11.8±4.3 fold increase in interaction with HOXA11-WT and a 13.7±3.6 fold increase with HOXA11-ΔH3 in the presence of DNA. DNAse treatment decreased the interactions to 6±0.24 and 7±1.1 for HOXA11-WT and -ΔH3 respectively. These data suggest the physical interaction between HoxA11 and Meis1b is a protein-protein association and that the presence of DNA may serve to stabilize this interaction. We next investigated the effects of HOXA11 on megakaryocytic differentiation. The human erythroleukemia cell line, K562, was stably transfected with expression vectors for either HOXA11-WT or -ΔH3. The transfected cell lines were then treated with the pharmacological agents, phorbol-12 myristate-13 acetate (PMA) or staurosporine (STSP), to induce megakaryocytic differentiation. Both agents induce surface expression of the megakaryocytic/platelet-specific antigen, CD61 and cause morphological changes. CD61 expression in untransfected cells increased from 1.6±0.4% at baseline to 91±0.5% with PMA and to 81±2.4% when treated with STSP. Marked differences in CD61 expression and viability were seen in transfected cells. HOXA11-WT and -ΔH3 lead to a significantly less CD61 expression, 53±10% and 69.8±5.8% respectively following PMA induction, compared to untransfected cells. PMA treatment of cells that constitutively express HOXA11-WT or -ΔH3 resulted in a marked reduction in cell viability and suggests that the normal silencing of HOXA11 in early hematopoiesis may be an essential aspect of its function. Neither HOXA11-WT nor -ΔH3 cells showed an increase in expression of CD61 expression following STSP treatment, 10.1±5.9% and 3.7±2.4% respectively. Interestingly, nearly all transfected cells treated with STSP remained viable, suggesting divergent signaling pathways are utilized by this differentiating agent. There were no substantial differences between wild type and mutant HOXA11 with either inducing agent, suggesting that inhibition of differentiation by HOXA11 involves other non-homeodomain regulatory sequences of this gene. Taken together these data begin to give us insight into the molecular mechanisms by which HoxA11 regulates megakaryocytic differentiation.

scholarly journals Discrimination of DNA binding sites by mutant p53 proteins.

1995 ◽  
Vol 15 (9) ◽  
pp. 5196-5202 ◽  
Author(s):  
S K Thukral ◽  
Y Lu ◽  
G C Blain ◽  
T S Harvey ◽  
V L Jacobsen
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Base Pair ◽  
Molecular Genetic ◽  
Full Length ◽  
Single Amino Acid ◽  
Wild Type ◽  
Binding Assays ◽  
Wild Type P53

Critical determinants of DNA recognition by p53 have been identified by a molecular genetic approach. The wild-type human p53 fragment containing amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA binding assays, and full-length human p53 was used for transactivation assays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically altered forms of a known consensus DNA site. This refinement indicates that p53 binds with high affinity to two repeats of PuGPuCA.TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A/T).(T/A) GPyPyPy. These results were further confirmed by transactivation assays of yeast by using full-length human p53 and systematically altered DNA sites. Dimers of the pentamer AGGCA oriented either head-to-head or tail-to-tail bound efficiently, but transactivation was facilitated only through head-to-head dimers. To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutions were made at several positions within the DNA binding domain of p53, and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gln or Arg, and Arg-283 to Gln bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentamer (PuGPuCA), respectively. Thus, we implicate these residues in amino acid-base pair contacts. Interestingly, mutant Cys-277 to Gln bound a consensus site as two and four monomers, as opposed to the wild-type p53 fragment, which invariably binds this site as four monomers.

scholarly journals The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation

2015 ◽  
Vol 112 (32) ◽  
pp. 10002-10007 ◽  
Author(s):  
Liang Chen ◽  
Farooq Rashid ◽  
Abdullah Shah ◽  
Hassaan M. Awan ◽  
Mingming Wu ◽  
...  
Keyword(s):  
P53 Protein ◽  
P53 Gene ◽  
P53 Mutation ◽  
Single Amino Acid ◽  
Human Lung Cancer ◽  
Rna Aptamer ◽  
Wild Type ◽  
Wild Type P53

p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice.

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