Download What is p53

Introduction
Between January 1993 and July 1996, more than 4300 papers have been published in which
the term "p53" appears in the title! This massive interest in a single protein is almost
unprecedented and reflects the central place of p53 in the regulation of cell number and the
frequency with which abnormalities of p53 occur in human tumours. p53 has been named
Molecule of the Year by the editor's of the journal Science (262, 1958-59), and the
"guardian of the genome" by many researchers. This protein plays a major role in the
transcription ("reading") of DNA, in cell growth and proliferation, and in a number of
metabolic processes. Because p53 suppresses abnormal cell proliferation (it acts like an
'emergency brake' in the cell cycle), it may represent an important mechanism for protection
against cancer. It also appears to be involved in programmed cell death, or apoptosis. When
a mutation in the p53 gene results in the substitution of one amino acid for another, p53 loses
its ability to block abnormal cell growth. Indeed, some mutations produce a p53 molecule that
actually stimulates cell division and promotes cancer. Almost 50% of human cancers contain
a p53 mutation -- including cancers of the breast, cervix, colon, lung, liver, prostate, bladder,
and skin -- and these cancers are more aggressive, more apt to metastasize, and more often
fatal.
Structure
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phosphoprotein of about 390 amino acids which can be subdivided into four domains:
a highly charged acidic region of about 75 to 80 residues, a hydrophobic proline-rich
domain (position 80 to 150), a central region (from 150 to about 300), and a highly
basic C-terminal region
The sequence of p53 is well conserved in vertebrate species, but there have been no
proteins homologous to p53 identified in lower eucaryotic organisms
human gene: TP53, 17p13.1; mouse gene: Trp53; Clam (Mya arenaria) gene: ask
Charles W. Walker
SWISS-PROT entry for p53: protein sequence, 3D structure, domains, other links
Comparisons of the amino-acid sequence of human, African green monkey, golden
hamster, rat, chicken, mouse, rainbow trout and Xenopus laevis p53 protein indicated
5 blocks of highly conserved regions, which coincide with the mutation clusters
found in p53 in human cancers
evolution: Xenopus (68% homology)
picture: p53 interacting with DNA. Method: X-ray diffraction.
[See Cho Y et al. for details!].
The DNA (blue) and core domain (turquoise) are shown with the zinc atom
(red), with the position of the six hot spot amino acid residues (yellow).
Mutations in hot spot amino acids either interfere with protein-DNA contacts,
or disrupt integrity of the domain. Thus, all naturally occurring mutations in
p53 directly or indirectly affect the interaction of p53 with DNA,
demonstrating that sequence-specific DNA binding is central to the normal
functioning of p53 as a tumor suppressor.
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3D structure of the p53 core domain-DNA complex
Post-translational modifications
 p53 is phosphorylated at many sites (by casein kinase I and II, JNK1, cdk's, DNA-PK)
Expression
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The p53 protein is located in the nucleus of cells and is very labile. Agents which
damage DNA induce p53 to become very stable by a post-translational mechanism,
allowing its concentration in the nucleus to increase dramatically [Ref.]
Cellular functions
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suppresses progression through the cell cycle in response to DNA damage, thereby
allowing DNA repair to occur before replicating the genome; hence, p53 prevents the
transmission of damaged genetic information from one cell generation to the next
initiates apoptosis if the damage to the cell is severe (this protects the organism from
the growth of damaged cells, and so loss of p53 function is a key step in the neoplastic
cascade). Mediators of this effect: bax [Ref.]
often as a tumour suppressor: Mutations in p53 can cause cells to become
oncogenically transformed, and transfection studies have shown that p53 acts as a
potent transdominant tumour suppressor, able to restore some level of normal
growth to cancerous cells in vitro (!)
p53 is a potent transcription factor and once activated, it represses transcription of
one set of genes (several of which are involved in stimulating cell growth) while
stimulating expression of other genes involved in cell cycle control
the p53 pathway (picture summarizing the cellular functions of p53, 996, by Hall PA
et al.)
Metabolism
Involvement in diseases
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Mutations in the p53 gene are the most frequently observed genetic lesions in human
cancers!!
The function of p53 is critical to the way that many cancer treatments kill cells since
radiotherapy and chemotherapy act in part by triggering cell suicide in response to
DNA damage. This successful response to therapy is greatly reduced in tumours
where p53 is mutant so these tumours are often particularly difficult to treat
the tumor-derived mutant p53 proteins that have been tested thus far do not bind to
DNA in the same manner as wild-type p53
p53 works as an emergency brake on cancer development by killing cells that attempt
to proliferate in oxygen-deficient regions of tumors (cells with mutant p53 can
survice these conditions)
Applications
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mutant adenovirus killing cells lacking p53 in mouse model (human cervical
carcinomas grown in nude mice, clinical trials began in April 1996) (R1-R2-R3-R4)
clinical trials of p53 gene therapy in lung cancer successful (Nature Medicine 2,
974/985, Sep 1996) (R1)
Antibodies
Antibody Immunogen
Species Ig
Epitope
specific. Subclass
PAb 240
murine p53
Human,
Monkey,
Cow,
Murine,
Bird
PAb 246
-
-
Additional
information
Source
middle
(a.a.
210226)
mutant/wild type
Neomarkers
recogn. (denatur.),
Boehringer
only mutant type
BIODESIGN
when non-denatur.
BIOMOL
cond.
1
IgG1
-
BIOMOL
wild type only
BIODESIGN
1
IgG1
Immunohistochemical
detection of p53 with
NPAb 1801 in human
terminus Neomarkers
breast cancers
(a.a.32- BIODESIGN
correlates with the
79)
clinical outcome of
the disease
-
-
IgG1
PAb
1801
human p53bHuman
galactosidase
specific
fusion
protein
BP53-12
recombinant
Human
human wildspecific
type p53
IgG2a
NNeomarkers formalin-fixed,
terminus
BIOMOL
paraffin-embedded
(a.a. 20BIODESIGN tumor tissue section
25)
UB94-4
human
recombinant
primate
wild-type
p53 protein
IgG2
?
B20.1
human
recombinant human
p53 protein
IgG2a
NBiomeda
terminus
UBI
-
wild type and mutant;
does not react with
normal fibroblasts;
data sheets on-line
 for additional monoclonals, see Neomarkers, BIOMOL and Calbiochem
 In-depth information about p53 antibodies can be found at Soussi's p53 page
Purification
Ref.
Manipulation
 Temperature-sensitive mutants
Animal Models
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Entry for p53 in: FBS knockout database
p53-null mice with germline homologous exon 5 deletions were of normal weight and
litter size indicating that the absence of p53 had no adverse effect on pre-natal survival
and development in these mice. However, p53-null mice had a much higher
frequency of developing tumours than their wild-type counterpart
engineered virus kills tumor cells lacking p53 in promising mouse model for cancer
therapy (see here)
Open Questions
Contacting Researchers
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Jill Bargonetti-Chavarria (DNA binding, cell cycle)
Anindya Dutta (DNA replication, p21, cancer)
Peter A Hall (n vivo systems, p53 phylogeny)
Paul Jackson (email: [email protected]) (transcription)
David Lane (restoring p53's functions)
David Meek (phosphorylation)
Ola Myklebost (sarcoma, MDM2)
Moshe Oren (target genes, mdm2, induction of apoptosis, etc.)
Reisman lab p53 home page (p53 gene transcriptional regulation)
Varda Rotter (DNA binding, antigenic epitopes, immunoglobulins, splicing)
Thierry Soussi et al. (heterogeneity of p53 mutations, anti-p53 antibodies, extensive
p53 Web site!)
A more comprehensive list of p53 researchers may be found here
Web Resources
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Affymetrix launches GeneChip (c) p53 Assay
Scientific discussion: search and access newsgroups with DejaNews, or have a virtual
meeting in BioMOO (the biologist's virtual meeting place)
The 9th International p53 Workshop (May 9-13, 1998; Crete, Greece)
IARC p53 homepage: mutation database, introduction, links
p53 entry in the Tumor Gene Database
Human Gene Map at NCBI: short introduction, some links
p53 homepage (at Dundee)
p53 and leukemia (review in The Cancer Journal, full text!)
p53 Information Center (link list)
p53 page (a comprehensive collection of useful links, with descriptions!)
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p53 assay kits by Calbiochem
References for further reading
Recent reviews:
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Hansen, R. and Oren, M. p53; from inductive signal to cellular effect. Curr. Op. Gen.
Dev. 7: 46-51 (1997).
Ambs S. Interactive effects of nitric oxide and the p53 tumor suppressor gene in
carcinogenesis and tumor progression. FASEB J 11(6), 443-448 (1997)
Hall PA. Tumor suppressors: a developing role for p53? Curr Biol 7(3), R144-R147
(1997)
Other papers:
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Ko, L. and Prives, C. p53: puzzle and paradigm. Genes and Development 10: 10541072 (1996).
Marx J New link found between p53 and DNA repair [news; comment] Science 266:
1321-2 (1994)
Chiarugi V; Ruggiero M Role of three cancer "master genes" p53, bcl2 and c-myc on
the apoptotic process. Tumori 82: 205-9 (1996)
Hale AJ; Smith CA; Sutherland LC; Stoneman VE; Longthorne VL; Culhane AC;
Williams GT Apoptosis: molecular regulation of cell death. Eur J Biochem 236: 1-26
(1996)
Bosari S; Viale G The clinical significance of p53 aberrations in human tumours.
Virchows Arch 427: 229-41 (1995)
Chang F; Syrjanen S; Syrjanen K Implications of the p53 tumor-suppressor gene in
clinical oncology. J Clin Oncol 13: 1009-22 (1995)
Bogler O; Huang HJ; Kleihues P; Cavenee WK The p53 gene and its role in human
brain tumors. Glia 15: 308-27 (1995)
Lee JM; Bernstein A Apoptosis, cancer and the p53
What is p53 ?
After the identification of the p53 protein and the subsequent cloning of p53
genes from several species, early observations suggested that p53 may function
as an oncogene, because overexpression of p53 appeared to cause oncogenic
transformation of cells. In the late 1980s, however, several critical discoveries
defined the normal function of p53 to be anti-oncogenic. Wild-type p53 genes,
when introduced into cells, were found to be growth suppressive. The
screening of DNA from colon cancer patients revealed that p53 mutations
occur with unusually high frequency in tumor tissue, an observation that was
extended to most of the other major forms of human cancer. Indeed, members
of Li-Fraumeni cancer-prone families were shown to carry germ-line p53
mutations. The importance of these observations was underscored by the
finding that mice that are homozygous null for p53, although developmentally
competent, are highly predisposed to tumors.
The functional character of the p53 protein was determined by experiments
showing that p53 contains a strong transcriptional activation domain within
its amino terminus and that it is a tetrameric, sequence-specific DNA-biding
protein with a defined cognate binding site containing two copies of the 10-mer
(5'-RRRCA/TT/AGYYY-3'). Although the p53 protein acts as a
transcriptional activator of genes containing p53-binding sites, it is also
capable of strongly inhibiting transcription from many genes lacking p53binding sites. Several oncogenic DNA viruses express viral gene products that
associate with and inhibit the trans-activation function of p53, notably SV40
large T antigen, the adenovirus E1B 55-kD protein, and the E6 protein of
oncogenic forms of human papillomavirus (HPV E6). In cells, p53 can
associate with a 90-kD protein, identified as the product of the mdm-2
oncogene, which is amplified in some types of tumors. When bound to mdm-2,
p53 can no longer function as an activator of transcription.
p53 plays multiple roles in cells. Expression of high levels of wild-type (but not
mutant) p53 has two outcomes: cell cycle arrest or apoptosis. The observation
that DNA-damaging agents induce levels of p53 in cells led to the definition of
p53 as a checkpoint factor, akin, perhaps, to the product of the fad9 gene in
yeast. While dispensable for viability, in response to genotoxic stress, p53 acts
as an "emergency brake" inducing either arrest or apoptosis, protecting the
genome from accumulating excess mutations. Consistent with this notion, cells
lacking p53 were shown to be genetically unstable and thus more prone to
tumors.
Funkcie p53
Úvod:
Medzi januárom 1993 a júlom 1996 bolo publikovaných viac ako 4300 článkov,
v ktorých názve sa objavil proteín p53. Tento obrovský záujem o jediný proteín odráža jeho
dôležitú úlohu v regulácii počtu buniek a frekvenciu s akou sa abnormality p53 vyskytujú
v ľudských tumoroch. p53 bol mnohými vedcami označený ako „strážca genómu“.
Tento proteín hrá dôležitú úlohu pri transkripcii („čítaní“) DNA, pri raste bunky
a proliferácii, a v mnohých metabolických procesoch. Keďže p53 potláča abnormálnu
bunkovú proliferáciu (pôsobí ako „záchranná brzda“ v bunkovom cykle), predstavuje dôležitý
mechanizmus v ochrane proti vzniku nádorov. Má tiež dôležitú úlohu pri programovanej smrti
buniek, apoptóze.
Keď mutácia v géne p53 spôsobí náhradu jednej aminokyseliny za inú, p53 stratí svoju
schopnosť blokovať abnormálny rast bunky. Takáto mutácia má za následok vznik molekuly
p53, ktorá dokonca stimuluje bunkové delenie a podporuje vznik nádoru.
Takmer 50% ľudských nádorov obsahuje mutáciu p53 – vrátane nádorov prsníka,
krčku maternice, hrubého čreva, pľúc, pečene, prostaty, močového mechúra a kože – a tieto
nádory sú agresívnejšie, majú väčší sklon metastázovať a častejšie sa končia fatálne.
Štruktúra:
Fosfoproteín zložený z približne 390 aminokyselín, ktorý môže byť rozdelený na štyri
domény: kyselinový úsek o dĺžke 75-80 aminokyselinových zvyškov, hydrofóbna na prolín
bohatá doména (od 80 do 150), centrálny úsek (od 150 do 300) a vysoko zásaditý Cterminálny úsek.
Sekvencia p53 je dobre zachovaná u stavovcov, ale u nižších eukaryotických
organizmov proteín homologický s p53 identifikovaný nebol.
Ľudský gén TP53 sa nachádza na 17p13.1.
Porovnávaním aminokyselinovej sekvencie u človeka, africkej zelenej opice, zlatého
škrečka, potkana, sliepky, myši, dúhového pstruha a Xenopus laevis p53 vykázal 5 úsekov
vysokokonzervovaných oblastí, ktoré splývajú so zmutovanými zhlukmi nájdenými v p53
ľudských nádorov.
Expresia:
Proteín p53 je lokalizovaný v jadre bunky a je veľmi labilný. Činitele, ktoré
poškodzujú DNA, nútia posttranslačným mechanizmom p53 k stabilite, čo umožní zvýšenie
jeho koncentrácie v jadre bunky.
Bunkové funkcie:
Potláča progresiu cez bunkový cyklus ako odpoveď na poškodenie DNA, teda
umožňuje opravu DNA pred replikáciou genómu. Z tohto dôvodu p53 predchádza prenosu
poškodenej genetickej informácie z jednej generácie buniek na druhú.
Iniciuje apoptózu, ak je poškodenie bunky závažné (teda chráni organizmus pred
rastom poškodených buniek a tak strata funkcie p53 je kľúčovým krokom v neoplastickej
kaskáde).
Pôsobí ako tumor supresor: mutácia p53 môže zapríčiniť onkogénnu transformáciu
bunky. Štúdie transfekcie ukázali, že p53 pôsobí ako účinný transdominantný tumor supresor,
ktorý je schopný obnoviť určitú hladinu normálneho rastu buniek u kanceróznych buniek in
vitro.
p53 je účinný transkripčný faktor a aktivovaný potláča transkripciu jednej skupiny
génov (z ktorých mnohé sa zúčastňujú na stimulácii rastu buniek), zatiaľ čo stimuluje
expresiu inej skupiny génov, ktorá kontroluje bunkový cyklus.
Účasť na chorobách:
Mutácie v géne p53 sú najviac frekventovanými genetickými faktormi pozorovanými
u ľudských nádorov.
Funkcia p53 je dôležitá pri spôsobe, ktorým mnohé liečivá na rakovinu zabíjajú
bunky. Rádioterapia a chemoterapia účinkujú pri spúšťaní bunkovej samovraždy ako
odpovedi na poškodenie DNA. Táto úspešná odpoveď na terapiu je redukovaná pri tumoroch,
kde je zmutovaný p53, teda tieto tumory sú často obtiažne liečiteľné.
Doteraz testované zmutované proteíny p53 sa neviažu na DNA rovnakým spôsobom
ako normálny typ p53.
p53 pôsobí ako záchranná brzda pri rozvoji rakoviny zabíjaním buniek, ktoré sa
pokúšajú proliferovať v oblastiach tumoru s deficitom kyslíka (bunky so zmutovaným p53
môžu prežívať aj za takýchto podmienok).