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p53 Isoforms: Key Regulators of the Cell Fate Decision

January 2016
Cold Spring Harbor Perspectives in Medicine 6(8):a026039

DOI:10.1101/cshperspect.a026039

Authors:

Sebastien M. Joruiz

National Cancer Institute (USA)

Jean-christophe Bourdon

University of Dundee

It is poorly understood how a single protein, p53, can be responsive to so many stress signals and orchestrates very diverse cell responses to maintain/restore cell/tissue functions. The uncovering that TP53 gene physiologically expresses, in a tissue-dependent manner, several p53 splice variants (isoforms) provides an explanation to its pleiotropic biological activities. Here, we summarize a decade of research on p53 isoforms. The clinical studies and the diverse cellular and animal models of p53 isoforms (zebrafish, Drosophila, and mouse) lead us to realize that a p53-mediated cell response is, in fact, the sum of the intrinsic activities of the coexpressed p53 isoforms and that unbalancing expression of different p53 isoforms leads to cancer, premature aging, (neuro)degenerative diseases, inflammation, embryo malformations, or defects in tissue regeneration. Cracking the p53 isoforms' code is, thus, a necessary step to improve cancer treatment. It also opens new exciting perspectives in tissue regeneration.

. p53 isoform-specific small interfering RNAs (siRNAs)

…

. p53 isoform-specific antibodies

…

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p53 Isoforms: Key Regulators of the

Cell Fate Decision

Sebastien M. Joruiz and Jean-Christophe Bourdon

Dundee Cancer Centre, University of Dundee, Ninewells Hospital and Medical School,

Dundee DD1 9SY, United Kingdom

Correspondence: j.bourdon@dundee.ac.uk

It is poorly understood how a single protein, p53, can be responsive to so manystress signals

and orchestrates very diverse cell responses to maintain/restore cell/tissue functions. The

uncovering that TP53 gene physiologically expresses, in a tissue-dependent manner, several

p53 splice variants (isoforms) provides an explanation to its pleiotropic biological activities.

Here, wesummarize a decade of research on p53 isoforms. Theclinical studies and the diverse

cellular and animal models of p53 isoforms (zebraﬁsh, Drosophila, and mouse) lead us to

realize that a p53-mediated cell response is, in fact, the sum of the intrinsic activities of the

coexpressed p53 isoforms and that unbalancing expression of different p53 isoforms leads to

cancer, premature aging, (neuro)degenerative diseases, inﬂammation, embryo malformations,

or defects in tissue regeneration. Cracking the p53 isoforms’ code is, thus, a necessary step to

improve cancer treatment. It also opens new exciting perspectives in tissue regeneration.

p53 is a central sensor of cell signals and a mas-

ter regulatorof cell response to damage (Lane

and Levine 2010). But how can so many distinct

extracellular and intracellular signals modulate

p53 activity? Howcan onlyone protein bind spe-

ciﬁcallyto so manydifferentDNA sequences( p53

response elements)(el-Deiry etal. 1992; Bourdon

et al. 1997; Khoury and Bourdon 2011; Simeo-

nova et al. 2012) and directly regulate expression

of thousands of genes? How does p53 select the

target genes to trigger on time the appropriatecell

responses to so many different cellular damages?

Why is it so difﬁcult to link TP53 mutation status

to prognosis and cancer treatment?

The TP53 gene is highly conserved in mul-

ticellular organisms (Lane et al. 2010). It is lo-

cated on human chromosome 17p13.1 and is

composed of 13 exons of which the ﬁrst is non-

coding (Fig. 1A). TP53 contains the Hp53Int1

and WRAP53 genes within exon-1/intron-1

(Reisman et al. 1996; Mahmoudi et al. 2009).

It presents multiple genetic polymorphisms de-

ﬁning more than 100 distinct TP53 haplotypes,

some of which are correlated with an increased

risk of cancer (Dumont et al. 2003; Garritano

et al. 2010; Wu et al. 2013). Although it is un-

equivocally established that TP53 is the most

frequently mutated gene in human cancer, it is

still difﬁcult in the clinic to link TP53 mutation

status to cancer treatment and clinical outcome,

suggesting that the p53 pathway is not entirely

understood. The discovery that the TP53 gene

encodes several different splice variants may ex-

plain the discrepancy.

Editors: Guillermina Lozano and Arnold J. Levine

Additional Perspectives on The p53 Protein available at www.perspectivesinmedicine.org

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Δ40p53γ

Δ40p53α

Associated polymorphisms

Polymorphisms affecting p53

internal promoter activity

G-Quadruplex DNA structure

Hp53Int1 gene

P2 internal promoter

WRAP53 gene

promoters

Polymorphism: g12718a

missense V217M

Polymorphism: c14007g

missense T312S

Polymorphism: g16970c

missense G360A

si133a/b

109

8765432

iii

vii

viii

p53/Δ40 Δ133/Δ160

c11455g

(P72R)

t12081c (intronic)

g12247a (intronic)

g12273a (intronic)

g11338a (silent)

c11298a (intronic)

ic)

PIN3 16 bp duplication

acc tggagggctg ggg (1268–1273)

t12239c (intronic)

(missense P47S)

Encoded protein:

Δ133p53γ or Δ160p53γ or

Δ133p53β or Δ160p53β or

Δ133p53α and Δ160p53α

Δ133p53α or Δ160p53α or

9β

9γ

9β

9βΔ40p53β

Δ40p53 only

Δ40p53β only

Δ40p53γ only

123 56 789

9β9γ10 11

siγ

siγsiα

siβ

siSplice

siE7/E8

siE7

siE2

p53γ or Δ40p53γ or p53γ and

p53β or Δ40p53β or p53β and

p53α or Δ40p53α or p53α and

Δ133p53γ and Δ160p53γ

Δ133p53β and Δ160p53β

Figure 1. TP53 locus and p53mRNAs. All introns/exons are represented to scale. Black boxes represent non-

coding sequences, whereas coding sequences are colored. (A) The human TP53 gene’s locus structure. The TP53

gene, which is composed of 11 exons and two cryptic exons (9band 9g), encodes several p53 isoforms

attributable to alternative promoters (BP1 and P2) and alternative retention of the cryptic exons. The non-

coding exon-1 and intron-1 contain different promoters for the WRAP53 gene (antisense-coded) and intron-1

contains the Hp53Int1 gene. A G-quadruplex DNA structure located within intron-3 modulates splicing of

intron-2 and activities of the internal p53 promoter P2. Several polymorphisms (including Pin3 and R72P)

change activities of the internal p53 promoter (P2). (B) p53 mRNAs. The TP53 gene encodes nine different

mRNAs attributable to the alternative promoters (BP1 and P2) and splicing (^). The promoter P1, located

upstream of exon-1, encodes for intron-2 spliced (i, ii, and iii) or intron-2 retained (iv, v, and vi) mRNAs. The

intron-2 spliced mRNAs can encode the full length (ATG1) and/or the D40 (ATG40) proteins, depending on the

cell context, whereas the mRNA retaining intron-2 can only encode the D40 proteins. The P2 initiation tran-

scription site is located in intron-4 and encodes for three transcripts (vii, viii, and ix), which encode the D133

and the D160 forms. Small interfering RNAs (siRNAs) targeting the different p53 isoforms are represented on

top of the corresponding exons or introns.

S.M. Joruiz and J.-C. Bourdon

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p53 splice variants were ﬁrst identiﬁed in the

late 1980s in human and mouse (Matlashewski et

al. 1984; Wolf et al. 1985). Thereafter, an alterna-

tive splicing of TP53 intron-9 has been described

(Arai et al. 1986; Flaman et al. 1996). To date, in

human, nine p53 mRNAs (Fig. 1B) encoding 12

different p53 protein isoforms have been des cribed

(Bourdon et al. 2005; Marcel et al. 2010a), p53a

(also named full-length p53, FLp53, canonical

p53, TAp53a), p53b(or p53i9), p53g,D40p53a

(or DNp53, p44 or p47), D40p53b,D40p53g,

D133p53a,D133p53b,D133p53g,D160p53a,

D160p53b,andD160p53g(Fig. 2A).

THE HUMAN p53 ISOFORMS

For decades, it was thought that one gene en-

codes one protein. The sequencing of the human

genome changed this dogma and revealed that

98% of the human genes are alternatively

spliced and contain multiple initiation sites of

transcription (promoter). The TP53 gene is no

exception. To date, it is reported that human

TP53 differentially expresses in normal tissue at

least nine mRNAs in a tissue-dependent manner.

They are a result of alternative promoter usage

(P1 and P2) and alternative splicing of intron-2

and intron-9 (Fig. 1B). Furthermore, depending

on the cell type, the translation of the p53

mRNAs can be initiated at different codons.

For the mRNAs transcribed from the proximal

promoter (P1), translation can be initiated at

codons 1 and/or 40, whereas the mRNAs tran-

scribed from the internal promoter (P2) trans-

lation can be initiated at codons 133 and/or 160.

The fully spliced p53 transcript (i) encodes

the canonical p53 protein (p53a) but also en-

codes the D40p53aisoform thanks to an inter-

nal ribosomal entry site (IRES) (Yin et al. 2002;

Candeias et al. 2006; Ray et al. 2006). This tran-

script also exists with two different alternative

splicings of exon-9 retaining thus the exon-9b

or -9g(ii/iii) and encoding, respectively, the

p53band/or D40p53b, and the p53gand/or

D40p53g. Both exon-9band exon-9gcontain

stop codons so that exon-10 and -11 are non-

coding in band gp53 mRNA splice variants.

The second type of transcript, also expressed

from the promoter P1, conserves intron-2

(iv/v/vi). Retention of this intron leads to sev-

eral stop codons when translation is initiated

from codon 1, thus preventing synthesis of full-

length p53 proteins. However, translation can

still be initiated at codon 40 so that this group

of mRNAs encodes exclusively D40p53a,

D40p53b,orD40p53g. The mRNAs vii/viii/ix

are transcribed from the internal promoter P2

located within intron-4. Their translation can

start either at the codons 133 or 160 encoding,

thus, the D133 and D160 p53 protein isoforms

(a,b,org, respectively).

At the protein level, p53a(canonical p53)

was the ﬁrst p53 protein isoform to be identi-

ﬁed. It is a 393-amino-acid-long protein with

seven functional domains (Fig. 2A,B). The ami-

no terminus is composed of two transactivation

(TA) domains, TA-1 and TA-2, which are re-

quired to induce a distinct subset of p53-target

genes. p53aalso contains a proline-rich domain

(PRD), a DNA-binding domain (DBD), and a

hinge domain (HD). The carboxyl terminus is

composed of an oligomerization domain (OD)

and a negative regulation domain (a). The neg-

ative regulation domain, rich in lysine (K-rich),

undergoes many different posttranslational

modiﬁcations (phosphorylation, methylation,

acetylation, sumoylation, ubiquitinylation, ned-

dylation, etc.), which regulate p53aactivity and

stability (Meek and Anderson 2009). In addi-

tion, the hinge and adomains contain several

nuclear localization signals (NLSs). The p53

DBD protein sequence is highly conserved

through evolution. It contains several conserved

cysteines and histidines that coordinate Zn

2þ

essential for p53 conformation and DNA-

binding activity (Fig. 2B) (Pavletich et al. 1993;

Cho et al. 1994; Xue et al. 2009).

The D40, D133, and D160 protein isoforms

are, respectively, lacking the ﬁrst 39, 132, and

159 amino acids. The D40 isoforms have lost

the TA-1 but still retain the TA-2 and the com-

plete DBD. The D133 isoforms lack both TA

domains and a small part of the ﬁrst conserved

cysteine box of the DBD, whereas the D160 iso-

forms lack both TA domains and the entire

ﬁrst conserved cysteine box of the DBD, retain-

ing the three other conserved cysteine boxes of

the DBD.

From the TP53 Gene to p53 Protein Isoforms

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356 7810 11i44i2

p53α

TA-1

TA-2

TA-2 PRD

PRD

SAPUSAPUSAPU

CM1

DNA-binding domain

HD OD

ODα

p53γ

p53β

Δ40p53α

Δ40p53β

Δ133p53β

Δ160p53β

Δ40p53γ

Δ133p53γ

Δ160p53γ

Δ133p53α

Δ160p53α

393

331

300

240

180

120

9β

9γ

332

332 341

346

332

IRGRERFEM

MEEPQSDPSV

DEAPRMPEAA PRVAPAPAAP TPAAPAPAPS WPLSSSVPSQ KTYQGSYGFR LGFLHSGTAK

SVTCTYSPAL NKMFCQLAKT CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE

EPPLSQETFS DLWKLLPENN VLSPLPSQAMDDLMLSPDDI EQWFTEDPGP

DQTSFQKENC

MLLDLRWCYFLINSS

FRELNEALEL KDAQAGKEPG GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD

Conserved regions

TIHYNYMCNS

VGSDCT

RCSDSDGLAP PQHLIRVEGN LRVEYLDDRN TFRHSVVVPY EPPE

PGSTKRALPN NTSSSPQPKK KPLDGEYFTL Q β

SCMGGMNRRP KKGEPHHELPDRRTEEENLREVRVCACPGR

SGNLLGRNSF

ILTIITLEDS

140

160133

NLS

Oligomerization domain

GEYFTLQ

GEYFTLQIRG...AG KE...HSS H...KKGQSTSR...DSD

DQ TSFQKENC

MLLDLRWCYFLINSS

GEYFTLQ

DO-12

KDa

3933573252919462401

421/BP53.10

DO-1 1801

DO-7 DO-11 240

9β9γ9

CM1

Figure 2. Human p53 protein isoforms. All exons and domains are represented to scale. (A) Schematic of the 12

p53 isoforms. The color of the protein domains matches the corresponding exon. p53ais composed of two

transactivation domains (TA-1 and TA-2), a proline-rich domain (PRD), the DNA-binding domain (DBD), the

hinge domain (HD), the oligomerization domain (OD), and the aregulatory domain. Compared with p53a,

D40 forms lack the TA-1 because of alternative translation initiation at ATG40. The D133 and D160 protein

isoforms are transcribed from P2 and lack TA-1, TA-2, PRD, and part of the DBD. Regarding the carboxy-

terminal isoforms, band gforms are attributable to an alternative splicing of intron-9 (brown and pink). As they

contain the entire exon-9, they encode the beginning of the OD. The theoretical molecular weight of each protein

isoform is indicated. The epitope boundaries of different p53 antibodies are represented by dotted lines. It is

important to note that Sapu and CM1 polyclonal antibodies contain multiple epitopes within the ﬁrst 80 amino

acids of p53aand several epitopes in the a-carboxy-terminal regulatory domain, explaining the enhanced

detection of p53acompared with the other p53 protein isoforms. (B) Amino-acid sequence of human p53

isoforms. The color of the amino-acid sequence matches the encoding exon. The translation initiation methi-

onines (M) are indicated in red. The a,b,orgpeptide sequences are indicated. The underlined amino-acid

sequences relate to the conserved cysteine domains in the DBD. Three nuclear localization sequences (NLS) are

represented in black boxes. One is common to all isoforms (exon-9) and two are speciﬁc of the aforms. The OD

encoded by exon-9 and -10 is shown in a red box.

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All p53 isoforms, whether transcribed from

the P1 or P2 promoter, can alternatively splice

exon-9band -9gto produce the b- and g-car-

boxy-terminal protein domains, replacing part

of the oligomerization domain and the entire

adomain. The b-carboxy-terminal protein is

composed of 10 amino acids, although the

g-carboxy-terminal protein domain is com-

posed of 15 amino acids. Of note, the ﬁrst seven

amino acids of the oligomerization domain are

present in the a,b, and gp53 isoforms.

SCIENTIFIC TOOLS TO INVESTIGATE

p53 ISOFORM BIOLOGICAL ACTIVITIES

(RT-q)PCR

The most reliable method to identify and quan-

tify p53 splice variants is (RT-q)PCR (quanti-

tative reverse transcription polymerase chain

reaction). Several protocols and a set of primers

have been designed and published to detect and

quantify p53 isoform mRNAs in cell lines and

tumor samples (Khoury et al. 2013). However,

there is not always a strict correlation between

the expression levels of p53 mRNAvariants and

p53 protein isoforms because p53 protein iso-

forms are also regulated at the posttranscrip-

tional level.

Small Interfering RNAs (siRNAs)

We and others have developed several siRNAs

and shRNAs targeting speciﬁcally distinct exons

or introns of human TP53, which enable knock-

down differentially and speciﬁcally subsets of

human p53 splice variants (Fig. 1B; Table 1).

The siRNAs are essential tools to determine

the biological activities of endogenous p53 pro-

Table 1. p53 isoform-speciﬁc small interfering RNAs (siRNAs)

Position on

sequence (nt)

GenBank

accession number

(NG_017013) Exon location Exon sequence Targeted forms

si E2 15968–15986 Exon 2 GCAGUCAGAUCCUAGCGUC p53a, p53b, p53g,

D40p53a,D40p53b,

D40p53g

si 133a 17212–17230 Intron 4 GGAGGUGCUUACACAUGUU D133p53a,D133p53b,

D133p53g,D160p53a,

D160p53b,D160p53g

si 133b 17271 –17289 Intron 4 CUUGUGCCCUGACUUUCAA D133p53a,D133p53b,

D133p53g,D160p53a,

D160p53b,D160p53g

si E7 18322–18340 Exon 7 GCAUGAACCGGAGGCCCAU All

si E7/E8 18363–18370/

18714–18724

Exon 7 and 8 GACTCCAGTGGTAATCTAC All

si b19211–19229 Exon 9bGGACCAGACCAGCUUUCAA p53b,D40p53b,

D133p53b,D160p53b

si g19009–19016/

19285–19295

Exon 9 and 9gCCCUUCAGAUGCUACUUGA p53g,D40p53g,

D133p53g,D160p53g

si Splice 19284–19302 Exon 9band 9gGAUGCUACUUGACUUACGA p53b,D40p53b,

D133p53b,D160p53b,

p53g,D40p53g,D

133p53g,D160p53g

si a21846–21864 Exon 10 GUGAGCGCUUCGAGAUGUU p53a,D40p53a,

D133p53a,D160p53a

From the TP53 Gene to p53 Protein Isoforms

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tein isoforms (Bourdon et al. 2005; Fujita et al.

2009; Aoubala et al. 2011; Terrier et al. 2011,

2012, 2013; Marcel et al. 2012, 2014; Wei et al.

2012; Bernard et al. 2013; Mondal et al. 2013).

Antibodies

Several mouse monoclonal antibodies are avail-

able to detect endogenous p53 protein isoforms

by immunoﬂuorescence, immunohistochem-

istry (IHC) on parafﬁn-embedded tissue, and

western blotting (Marcel et al. 2013). The epi-

tope of each p53 antibody has been determined

by ELISA on an epitope-mapping peptide li-

brary (Figs. 2A, 3B; Table 2). The mouse mono-

clonal antibodies DO-1 and DO-7 recognize the

same epitope within the TA-1 domain and can

only detect p53a, p53b, and p53g. The mouse

monoclonal antibody 1801 recognizes an epi-

tope within the TA-2 domain and, thus, detects

p53a, p53b, p53g,D40p53a,D40p53b, and

D40p53g. In some cell lines and tissue, 1801

cross-reacts with a protein not related to p53.

The mouse monoclonal 421 and BP53.10 rec-

ognize a similar epitope within the adomain

and, thus, detect p53a,D40p53a,D133p53a,

and D160p53a. The other mouse monoclonal

antibodies (DO-11, 240, DO-12, and 1620) rec-

ognize distinct epitopes within the DBD and

should, thus, theoretically detect all p53 pro-

tein isoforms. Because p53 protein isoforms

can be modiﬁed by posttranslational modiﬁca-

tions, which alter their migration on SDS-PAGE

(sodium dodecyl sulfate polyacrylamide gel

electrophoresis), it is recommended to transfect

cells with siRNA targeting distinct p53 splice

variants to validate that the bands detected

are p53 protein isoforms (Marcel et al. 2013).

Table 2. p53 isoform-speciﬁc antibodies

Epitope

Amino acids Exon Sequence Recognized forms

DO-1 20–25 2 SDLWKL p53a, p53b, p53g

DO-7 20–25 2 SDLWKL p53a, p53b, p53g

1801 46–55 4 SPDDIEQWFT p53a, p53b, p53g,

D40p53a,D40p53b,

D40p53g

1620 145–157 þ

201–212

5/6 LWVDST PPPGTRV þLRVEYLDDRN TF ND

DO-11 181–190 5/6 RCSDSDGLAP All

240 211–220 6 TFRHSVVVPY All

DO-12 256–267 7/8 TLEDSSGNLLGR All

SAPU 11–65/

290–301/

363–393

2/3/4/

8/9/

10/11

EPPLSQ-DPGPDEAPR/RKKGEPHHELPP/

RAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD

All

CM1 1–90/

379–393

2/3/

4/11

MEEPQSDPSV......TPAAPAPAPS/

RHKKLMFKTEGPDSD

p53a, p53b, p53g,

D40p53a,D40p53b,

D40p53g,D133p53a,

D160p53a

421 372–379 11 KKGQSTSR p53a,D40p53a,

D133p53a,

D160p53a

BP53.10 374–378 11 GQSTS p53a,D40p53a,

D133p53a,D160p53a

ND, not determined.

S.M. Joruiz and J.-C. Bourdon

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Furthermore, posttranslational modiﬁcations

within epitopes can prevent binding of p53

antibodies. Detection by DO-1 is impaired

by phosphorylation of serine-20 (Craig et al.

1999). Similarly, it is well established that bind-

ing of 421 antibody is prevented by posttrans-

lational modiﬁcation of lysine-372, serine-376/

378, threonine-377, and histidine-380 (Pospı

´si-

lova

´et al. 2000). The antibody BP53-10 could

be used to circumvent this problem as it is less

sensitive to posttranslational modiﬁcations sur-

rounding its epitope.

The rabbit (CM1) and sheep (SAPU) poly-

clonal antibodies have been raised against bac-

terially produced recombinant human p53a.

They recognize several distinct epitopes within

the amino-terminal and adomains so that

the detection of p53ais strongly enhanced

compared with the other p53 isoforms, thus

preventing any expression-level comparisons

between p53aand the other isoforms. CM1

and SAPU antibodies have a lower afﬁnity for

the D133/D160 and b/gforms that lack the

amino terminus or car boxyl-terminus epitopes,

respectively. Only SAPU recognizes an epitope

within the HD, which is common to all p53 pro-

tein isoforms. It can, thus, detect D133p53b/g

and D160p53b/g, whereas CM1 cannot.

THE p53 ISOFORM RELEVANCE IN CLINIC

In recent years, numerous studies have shown

that p53 isoforms are abnormally expressed in

breast cancer, melanoma, renal cell carcinoma

(RCC), acute myeloid leukemia (AML), colon

carcinoma, head and neck tumors (HNSSCs),

hepatic cholangiocarcinoma, ovarian cancer,

lung tumor, and glioblastoma (Bourdon et al.

2005, 2011; Anensen et al. 2006; Boldrup et al.

2007; Avery-Kiejda et al. 2008; Marabese et al.

120

180

240

300

331

393

1801

1620

1620 240

BP53.10421

DO-12

DO-1/DO-7

DO-11

9β

9γ

332

332 341

367

346

332

P2 γ

siγsiαsiβ

siSplice

siE7/E8

siE7

siE2

356 7899β9γ10 11i44i2

si133a/b

SCMGGMNRRP

IRGRERFEM

PGSTKRALPN

MEEPQSDPSV

DEAPRMPEAA PRVAPAPAAP TPAAPAPAPS WPLSSSVPSQ KTYQGSYGFR LGFLHSGTAK

SVTCTYSPAL NKMFCQLAKT CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE

EPPLSQETFS DLWKLLPENN VLSPLPSQAM DDLMLSPDDI EQWFTEDPGP

NTSSSPQPKK KPLDGEYFTL Q

DQTSFQKENC

MLLDLRWCYFLINSS

FRELNEALEL KDAQAGKEPG GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP

KKGEPHHELPDRRTEEENLREVRVCACPGR

SGNLLGRNSF

DSD

ILTIITLEDS

TIHYNYMCNS

VGSDCT

RCSDSDGLAP PQHLIRVEGN LRVEYLDDRN TFRHSVVVPY EPPE

Figure 3. Localization of the human p53 small interfering RNA (siRNA) and p53 antibodies’s epitopes. (A) p53

isoform-speciﬁc siRNAs are represented on the human TP53 gene. (B) p53 antibodies’ epitopes on human p53

protein. The underlined amino-acid sequences correspond to the epitopes of the named antibodies (mAb1801,

mAb240, mAb DO-11, and mAb DO-12). mAb DO-1 and DO-7 have the same epitope. mAb1620 antibody has

a conformational epitope that is represented by green boxes. The mAb 421 epitope is shown in a red box, whereas

the mAb BP53.10 epitope is underlined.

From the TP53 Gene to p53 Protein Isoforms

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2008; Fujita et al. 2009; Song et al. 2009; Hof-

stetter et al. 2010; Nutthasirikul et al. 2013;

Takahashi et al. 2013).

Several studies investigated whether p53

isoforms were associated with cancer patient

prognosis. First, it was reported that the evolu-

tion from colorectal adenoma to carcinoma

was potentially driven by an imbalance of

p53b/D133p53aratio in favor of the latter (Fu-

jita et al. 2009). In AML, distinct p53 isoform

biosignatures correlate with clinical outcome

˚nensen et al. 2012), whereas in cholangio-

carcinoma, down-regulation of the TAp53

isoforms (p53a/p53b/p53g) combined with

up-regulation of the D133 forms is associated

with a shortened overall survival (Nutthasirikul

et al. 2013).

Importantly, several studies in different hu-

man cancers have reported that the prognostic

value of TP53 mutation status is improved when

combined with p53 isoform expression. Wild-

type (WT) TP53 mucinous or serous ovarian

cancer patients expressing D40p53ahave a bet-

ter clinical outcome than WT TP53 mucinous

or serous ovarian cancer patients not expressing

D40p53a. Reciprocally, mutant TP53 serous

ovarian cancer patients expressing D133p53a

have a better disease-free and overall survival

than mutant TP53 serous ovarian cancer pa-

tients not expressing D133p53a(Hofstetter et

al. 2011, 2012; Chambers and Martinez 2012).

Furthermore, in WT TP53 ovarian cancers,

p53bexpression is associated with higher risk

of recurrence because it is a marker of serous

and poorly differentiated cancer (Hofstetter

et al. 2010).

In breast cancer, it was reported that p53b

expression is associated with smaller tumors

and longer disease-free survival in mutant

TP53 tumors, whereas D40p53 isoforms expres-

sion was found to be associated with the aggres-

sive triple negative subtype (Avery-Kiejda et al.

2014). In another study, it was shown that

mutant TP53 breast cancer patients expressing

p53ghave as a good prognosis as WT TP53

breast cancer patients, whereas mutant TP53

breast cancer patients devoid of p53gexpres-

sion have a particularly poor prognosis (Bour-

don et al. 2011).

Altogether, it suggests that the prognostic

values of p53b, p53g,D40p53a,o

rD133p53a

depend on the TP53 mutations status and the

cancer type.

Other studies have investigated whether p53

isoforms play active roles in cancer formation

and treatment. Silent TP53 mutation or muta-

tions in noncoding regions, such as IRES se-

quences, introns, or splicing sites, are associated

with cancer formation probably because they

lead to unbalanced p53 isoform expression de-

spite expressing WT p53 proteins (Hofstetter

et al. 2010; Grover et al. 2011; Khan et al.

2013). Furthermore, the pathogenic bacteria

Helicobacter pylori has recently been shown to

induce expression of D133 and D160 isoforms

in gastric epithelial cells, increasing their sur-

vival and probably promoting cancer formation

(Wei et al. 2012). Moreover, p53 isoforms are

involved in response to chemotherapy in vivo in

AML and melanoma (Anensen et al. 2006;

Avery-Kiejda et al. 2008; A

˚nensen et al. 2012).

In conclusion, p53 isoforms expression is

associated to clinical outcome of cancer pa-

tients. For a given cancer type, the accuracy of

patient prognosis is greatly improved by com-

bining p53 isoforms expression and TP53 mu-

tation status, which may be used to predict

response to treatment for some cancer types.

ANIMAL MODELS: IN SEARCH OF THE

PHYSIOLOGICAL RELEVANCE OF

ISOFORMS

The structure of the TP53 gene is highly con-

served through evolution (Bourdon et al. 2005;

Chen et al. 2005). Several p53 isoform animal

models were, thus, generated to investigate their

biological relevance and roles in carcinogenesis.

Zebraﬁsh

The zebraﬁsh TP53 gene (Zp53) has a dual gene

structure as its human counterpart (Storer and

Zon 2010). In addition to the Zp53aprotein,

corresponding to the human p53a(Cheng et al.

1997), the gene also encodes Zp53b,ZDNp53a,

and ZD113p53a. In addition, in some zebraﬁsh

strains, the Zp53 gene expresses from the inter-

S.M. Joruiz and J.-C. Bourdon

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nal promoter TA2Zp53, TA3Zp53, TA4Zp53,

and TA5Zp53 because of a polymorphism

(Fig. 4A).

ZDNp53ais produced through retention of

intron-2, similarly to the human D40p53 forms.

However, ZDNp53ais not completely identical

to the human form as the translation is initiated

in intron-2 and substitutes the 38 ﬁrst amino

acids of canonical p53 by 33 different amino

acids constituting a different TA domain (Da-

vidson et al. 2010). ZDNp53aaccumulates in

response to g-ray and is able to form a pro-

tein complex with Zp53athrough the OD.

When coexpressed with Zp53a, overexpres-

sion of ZDNp53ainduces developmental mal-

formations (hypoplasia, head malformation,

somites, and eyes). This phenotype does not

appear after overexpression of ZDNp53amutat-

ed in the oligomerization domain or in Zp53

morpholino-depleted embryo, suggesting that

ZDNp53aacts through oligomerization with

Zp53 isoforms during development.

Mp53

DTAp53

Zp53

ZΔNp53

ZΔ113p53

Zp53β

DΔNp53

TA-1 TA-2 DNA-binding domain C-ter

TA-1

TAD

DNA-binding domain

C-ter

1 123

138

61 113 260 275 373

368

260

294

187 388 495

385

41 87 218 286

242

361 390

349

381

i10

Mp53AS

MΔ41p53

Mp53Ψ

Ψ peptide:

β peptide:

AS peptide:

SKFAATLLKNILPYNRRVM

Mouse isoforms

Drosophila isoforms

Zebrafish isoforms

LQPRAFQALIKEESPNC

VTCSEVGSDFTWILCLPPGRL

Figure 4. p53 isoforms in animal models. Like human p53 protein isoforms, zebraﬁsh, Drosophila, and mouse

p53 proteins are also composed of several functional domains: the transactivation domain (TAD, yellow), the

DNA-binding domain (DBD, green) and the carboxy-terminal regulatory domain containing the oligomeri-

zation domain (C-ter, orange). For each species, all domains are represented to scale compared with the full-

length protein of the corresponding species. Hatched protein domains are encoded by cryptic exons within

introns (the corresponding intron is indicated). (A) Zebraﬁsh p53 isoforms. In the ZDNp53 isoform, the ﬁrst 33

amino acids are encoded bya cryptic exon in intron-2. In Zp53b, the last 19 amino acids are encoded by a cryptic

exon in intron-8. Of note, Zp53bis not homologous to human p53b.(B)Drosophila p53 protein isoforms. Like

in the human TP53 gene family, DDNp53 (also named Dp53) protein is encoded by an mRNA transcribed from

an internal promoter. DDNp53 is homologous to D40p53/D133p53/DNp63/DNp73 proteins. Only DTAp53

protein contains the conserved box I (FxxLW) corresponding to the transactivation domain (TA-1) of the p53

protein family. (C) Mouse p53 isoforms. In Mp53AS protein (alternatively spliced [AS]), the last 26 amino acids

of Mp53aare substituted by 17 others encoded by a cryptic exon in intron-10. Mp53AS protein is homologous

to human p53b.MD41p53 produced by alternative splicing of intron-2 is homologous to human D40p53a. The

Mp53Cis produced by alternative splicing of intron-6 adding 21 new amino acids as indicated. Up to now, no

homologous protein to Mp53Chas been found in other organisms or in human cells expressing the WT TP53

gene.

From the TP53 Gene to p53 Protein Isoforms

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ZD113p53ais homologous to human

D133p53a. It is produced from the internal pro-

moter, which is transactivated by Zp53a(Chen

et al. 2005; Marcel et al. 2010b). ZD113p53a

oligomerizes with Zp53ainhibiting apoptosis

by differentially modulating Zp53 target gene

expression (Ou et al. 2014). ZD113p53ainduc-

es expression of cell-cycle arrest genes, such as

p21 and cyclin-G

and of antiapoptotic genes,

such as Bcl-XL, while inhibiting expression of

proapoptotic genes like Reprimo or Bax (Chen

et al. 2009). Furthermore, ZD113p53ais strong-

ly induced after DNA double-strand break

(DSB) and activates the DNA –DSB repair path-

ways, notably by up-regulating the transcrip-

tion of repair genes such as RAD51, RAD52,

or lig4 (Gong et al. 2015).

Recently, Shi et al. reported that a natural

polymorphism, a 4-bp deletion within intron-

4, creates four new upstream translation initia-

tion sites in frame to the ZD113p53 open read-

ing frame. This leads to four protein isoforms

with a putative TA domain named TA2p53,

TA3p53, TA4p53, and TA5p53. They lack the

93 ﬁrst amino acids of Zp53athat are, respec-

tively, replaced by 65, 45, 18, and 9 amino acids

(Shi et al. 2015). These isoforms coexpressed

with Zp53aconfer resistance to irradiation. In

the same publication, Shi et al. also reported the

alternative splicing of Zp53 intron-8 leading to

Zp53b. However, Zp53bis not homologous to

human p53b.

Drosophila

The Drosophila melanogaster TP53 gene

(Dmp53) is the single Drosophila ortholog of

the mammalian p53 family and has a dual gene

structure with an internal promoter. It encodes

proteins homologous to p53, p63, and p73 (Lu

et al. 2009), and it is thought that, as the unique

member of the p53 family, it is likely that

Dmp53 carries the ancestral functions of all

p53 family genes. Dmp53 transcribes four differ-

ent mRNAvariants but, so far, only two p53 pro-

tein isoforms could be detected: DTAp53 and

DDNp53 (Fig. 4B). DTAp53 contains, in the ﬁrst

40 amino acids, the highly conserved TA domain

present in mammalian p53a(Bourdon et al.

2005). DDNp53 (also called Dp53), expressed

from the internal promoter, lacks the 123 ﬁrst

amino acids of DTAp53, which are replaced by

13 different ones encoded by a cryptic exon.

DDNp53 does not contain the conserved TA

domain but is still able to transactivate.

DDNp53 was the ﬁrst Drosophila p53 isoform

cloned and was thus called Dp53 as the investi-

gators thought they had cloned full-length Dro-

sophila p53 (Brodsky et al. 2000; Jin et al. 2000;

Ollmann et al. 2000). However, DDNp53 is ho-

mologous to the amino-terminally truncated

isoforms of the p53 family proteins (D40p53,

DNp63, or DNp73). The literature on Drosophila

p53 is, thus, confusing as most genetic studies do

not discriminate between DTAp53 and DDNp53

isoforms, reporting phenotype associated with

Dmp53 gene as being mediated by Dp53 protein.

Another source of confusion is that mutant

p53 ﬂies referred both to p53-null ﬂies and to

transgenic ﬂies overexpressing single-point mis-

sense mutant of Dmp53 (UAS-Dmp53R155H

or UAS-Dmp53H159N). However, p53-null

mutation leads to complete loss of p53 protein

expression and, thus, p53 activity, whereas mis-

sense mutation leads to overexpression of mu-

tant p53 proteins that are biochemically and

biologically very active as they directly interact

with numerous proteins modifying, thus, gene

expression and cell responses. p53-null muta-

tion can lead to different phenotypes than mis-

sense p53 mutation as shown in Drosophila and

mammalian models (Brodsky et al. 2000; Oll-

mann et al. 2000; Jassim et al. 2003; Lang et al.

2004; Olive et al. 2004; Wells et al. 2006; de la

Cova et al. 2014; Simo

´n et al. 2014)

The Dmp53 gene is involved in apoptosis

through the Reaper– Hid – Grim cascade by di-

rectly regulating Reaper expression in response

to damage (Brodsky et al. 2000, 2004). However,

its physiological activity is not limited to apo-

ptotic pathways. Recent studies unravel the pri-

mordial roles of Drosophila p53 proteinisoforms

in tissue regeneration by directly controlling

apoptosis-induced proliferation (AiP), com-

pensatory proliferation (CP), cell differentia-

tion, organ size, system growth, and cell compe-

tition (Peterson et al. 2002; Jassim et al. 2003;

Wells et al. 2006; Stieper et al. 2008; Mendes et al.

S.M. Joruiz and J.-C. Bourdon

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2009; Fan et al. 2010; Mesquita et al. 2010; Mo-

rata et al. 2011; Wells and Johnston 2012; de la

Cova et al. 2014; Simo

´n et al. 2014). This is con-

sistent with Dmp53 being involved in life-span

control (Bauer et al. 2005, 2010; Waskar et al.

2009; for review, see Martı

´n et al. 2009; Kashio

et al. 2014; Mollereau and Ma 2014).

However, the contribution of each Drosoph-

ila p53 protein isoform in those biological phe-

nomena is still poorly understood. Recently,

Dichtel-Danjoy et al. (2013) investigated the

respective roles of DTAp53 and DDNp53 over-

expression in apoptosis and AiP in the develop-

ing wing imaginal disc. AiP is a phenomenon in

which cells undergoing apoptosis after a stress

event (i.e., “undead cells”) secrete mitogens,

such as Wingless (Wg), Decapentaplegic (Dpp),

or Hedgehog (Hh) (homologous to human

Wnt, TGF-b, or Hedgehog, respectively) to pro-

mote proliferation of neighboring cells to re-

store the normal organ and body size. Thus, at

the same time that the stress event eliminates a

large number of cells, it also generates a stimu-

lus to proliferation. AiP is directly controlled by

Dmp53 (Wells et al. 2006; Dichtel-Danjoy et al.

2013; Simo

´n et al. 2014). Both DTAp53 and

DDNp53 protein isoforms were capable of acti-

vating apoptosis but they used different molec-

ular mechanisms. However, only DDNp53 (not

DTAp53) can induce Wg expression in “un-

dead” cells and enhanced proliferation of neigh-

boring cells in Dmp53-null ﬂies, suggesting that

DDNp53 is the main isoform that regulates AiP.

DTAp53 and DDNp53 seem, thus, to have dis-

tinct biological functions.

Mouse

The mouse TP53 gene (MsTP53) has a dual gene

structure as its human counterpart. To date,

four mouse p53 protein isoforms have been

published: Mp53a, Mp53AS, MD41p53a, and

Mp53C(Fig. 4C). Mp53ais the equivalent to

the canonical human p53a. Mp53AS is a car-

boxy-terminal splicing variant because of reten-

tion of part of intron-10. It encodes a 2-kDa

shorter protein with different carboxy-terminal

amino acids, which has a strong homology with

the human bp53 proteins. Mp53AS can bind

DNA and modulate p53-target-gene expression

in a promoter-dependent manner. Mp53AS can

induce apoptosis when expressed in absence of

Mp53a(Almog et al. 1997). However, when

Mp53AS and Mp53aare coexpressed, cells do

not induce apoptosis in response to damage

(Wolf et al. 1985; Almog et al. 2000). Mp53AS

and Mp53aoligomerize together and bind

DNA to regulate gene expression (Wu et al.

1994). Therefore, Mp53AS and Mp53aharbor

completely different/opposite activities when

they are expressed alone or in combination.

Recently, two knockin p53 mice, Mp53

D24

and Mp53

D31

-expressing carboxy-terminal

truncated mutants of p53 have been generated.

The Mp53

D24

mice contain a stop mutation at

codon 367 in the beginning of exon-11 so that

the mice express Mp53

D24

,MD41p53

D24

but

also the WT Mp53AS and Mp53Cproteins, be-

cause alternative splicing of intron-10 can still

occur (Hamard et al. 2013). The Mp53

D31

mice

contain a stop mutation at codon 360 at the end

of exon-10 so that the mice express Mp53

D31

MD41p53

D31

, and WT Mp53C. The Mp53

D31

mice do not express Mp53AS (Simeonova et al.

2013). In both studies, the MsTP53 gene has

been extensively sequenced to ensure the ab-

sence of any additional mutations in MsTP53

exons and introns. Interestingly, the Mp53

D24

and Mp53

D31

mice present different pheno-

types despite being of similar mixed genetic

background (BL6/129Sv). The Mp53

D24

and

Mp53

D31

mice have different blood cell counts

and different sizes of heart, thymus, spleen, tes-

tis, and cerebellum. Mp53

D31

mice also develop

oral leukoplakia (100%) and pulmonary ﬁbro-

sis (87%), traits not reported for Mp53

D24

mice.

One of the most intriguing differences is in the

repopulating capabilities of hematopoietic stem

cells (HSCs) from embryos of the Mp53

D24

and Mp53

D31

mice. WT matching mixed genet-

ic background mice (BL6/129Sv) were lethally

irradiated to destroy the bone marrow cells.

The irradiated mice were then transplanted

with HSCs from Mp53

D24

, Mp53

D31

,orWT

MsTP53 mice (BL6/129Sv). In both laborato-

ries, the transplantation of HSC from WT

MsTP53 mice rescued 100% of the irradiated

mice by regenerating the bone marrow, al-

From the TP53 Gene to p53 Protein Isoforms

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though none of the irradiated and nontrans-

planted mice survived, indicating that both

laboratories master this assay. Importantly, the

transplantation of HSC from Mp53

D31

mice

completely fails to repopulate the bone marrow

and to rescue, thus, the irradiated mice (0%

rescue, n¼5), whereas the transplantation of

HSC from Mp53

D24

mice rescue 70% (n¼6)

of the irradiated mice by repopulating the bone

marrow (Hamard et al. 2013; Simeonova et al.

2013).

Knowing the potent transcriptional activi-

ties of Mp53AS and its tissue-speciﬁc expres-

sion, it is likely that the different phenotypes

of the Mp53

D24

and Mp53

D31

mice are attribut-

able to differential expression of Mp53AS. Fur-

ther investigations will be required to determine

the physiological roles of Mp53AS in tissue re-

generation in mice.

Recently, an additional isoform attributed

to the alternative splicing of intron-6, Mp53C

(also named p53C) has been described. This

results from the physiological insertion of the

last 55 nucleotides of intron-6, which rapidly

leads to a stop codon. Mp53Cis thus a very

short p53 isoform containing the TA and pro-

line domains but no DBD (Senturk et al. 2014).

Mp53Cmay act as a prometastatic factor in

promoting epithelial–mesenchymal transition

and regulating mitochondrial activity togeth-

er with cyclophilin-D. Interestingly, Mp53C

seems to be expressed after damage in speciﬁc

stem cells during tissue regeneration in mice.

In liver injured with CCL4, the lesions get

smaller concomitantly to higher expression of

Mp53C. This is a fascinating result as it indi-

cates that the Msp53 gene is involved in tissue

regeneration in mammalians. Furthermore, it

opens new perspectives as p53 isoform expres-

sion could be manipulated to control organ re-

generation in mammalians.

Mp53CmRNA expression is unequivocal

in regenerating lung or liver mouse tissues; fur-

ther investigations will be required to conﬁrm

physiological p53Cexpression in WT human

cells. So far, endogenous human p53Cprotein

could only be detected in one cancer cell line

(Hop62) that bears a mutation in the acceptor-

splicing site of human TP53 intron-6. Such a

mutation abolishes the canonical splicing of in-

tron-6 and promotes the insertion of the last 49

nucleotides of human TP53 intron-6 leading to

a p53C-like protein as shown by the investiga-

tors (Senturk et al. 2014). The development of

human/mouse p53C-speciﬁc antibodies and

siRNAwould greatly help to assess p53Cexpres-

sion and study its biological activities.

TheMD41p53aisoform has also been genet-

ically investigated.MD41p53a(also named p44)

is the mouse counterpart of human D40p53a.

MD41p53aoverexpression in WT Mp53 mice

leads to a smaller animal with premature ag-

ing and shorter life span. These effects have

been attributed to abnormal insulin-like growth

factor (IGF) signaling, driving a reduced cellular

proliferation with increased senescence (Maier

et al. 2004; Gambino et al. 2013), inhibition of

embryonic stem cell (ESC) differentiation

(Ungewitter and Scrable 2010), neurodegenera-

tion (Pehar et al. 2010, 2014), and impaired

b-cell proliferation (Hinault et al. 2011), which

suggests that the MD41p53 transgenic mice

have altered tissue-regeneration capabilities.

To genetically investigate the biological ac-

tivities of D133p53 isoforms, Slatter et al. (2011)

have generated knockin mutant TP53 mice

that are deleted of exon-3 and -4 (MsD122p53),

expressing ubiquitously D133p53-like protein

(D122p53a) and probably D122p53AS in a

tissue-dependent manner because of alterna-

tive splicing of intron-10. The homozygote

MsD122p53 mice show enhanced proinﬂamma-

tory phenotype, features of autoimmune dis-

ease, and early tumor onset (Campbell et al.

2012; Sawhney et al. 2015). It was thus thought

that D122p53 proteins were oncogenic. How-

ever, it was recently genetically shown that

D122p53 proteins enhance the tumor-suppres-

sor activities of an attenuated p53 mutant delet-

ed of the proline domain (MDpro) (Slatter et al.

2015).

Altogether, the different animal models are

consistent in demonstrating that the p53 iso-

forms are potent and essential components of

the p53 pathway, being involved in cancer, ag-

ing, tissue regeneration, glucose metabolism,

embryo development, immune system, and

bacterial infection (Wei et al. 2012). The balance

S.M. Joruiz and J.-C. Bourdon

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between the different p53 isoforms is crucial in

determining cell-fate outcome.

REGULATION OF HUMAN p53 ISOFORM

EXPRESSION/ACTIVITIES

p53 isoform expression is regulated at the

transcriptional level by modulating the TP53

promoter activities and the alternative splicing

of intron-2 and intron-9.

In addition to the epigenetic events that reg-

ulate the tissue-speciﬁc activity of the p53 pro-

moters, the internal TP53 promoter activity is

also inﬂuenced by several polymorphisms, par-

ticularly the 16-bp insertion in intron-3 ( pin3)

and the R72P polymorphism in exon-4 altering

thus D133p53 isoform expression (Fig. 1A)

(Bellini et al. 2010; Marcel et al. 2012). In addi-

tion, the internal TP53 promoter is transacti-

vated by p53a, diverse p63/p73 isoforms, and

p68 (Moore et al. 2010; Aoubala et al. 2011;

Marcel et al. 2012).

SRSF1 and SRSF3, a highly conserved family

of splicing factors frequently deregulated in can-

cer, regulate the alternative splicing of TP53

intron-9 inhibiting retention of exon-9b/9g

(Tang et al. 2013; Marcel et al. 2014). Interest-

ingly, TG003, a small drug inhibitor of the Cdc2-

like kinases (Clks) that activate SRSF1 and

SRSF3, increases endogenous p53b/gexpres-

sion inhibiting cell proliferation in a p53 iso-

form-dependent manner (Marcel et al. 2014).

The alternative splicing of TP53 intron-2

leads to D40p53. Its expression is inﬂuenced

by a G-quadruplex structure within intron-3,

which is strengthened by the pin3 polymor-

phism that contains further G-quadruplex

structures (Marcel et al. 2011).

p53 isoform expression is also regulated at

the translational and posttranslational levels.

The D40p53 isoforms can be translated from

IRES controlled by IRES transactivating factors

(ITAFs), such as polypyrimidine-tract-bind-

ing protein (PTB), dyskerin, DAP5, annexin

A2, and PTB-associated splicing factor (PSF)

(Grover et al. 2008; Sharathchandra et al. 2012;

Weingarten-Gabbay et al. 2014).

Camus et al. (2012) have reported that all

p53 isoforms are ubiquitinated and degraded by

the proteasome. However, although MDM-2

binds to all p53 protein isoforms, it can only

promote the ubiquitination and degradation

of p53b. MDM2 binds probably D133p53 iso-

forms (a,b,org) through the DBD (Wallace

et al. 2006) and/or the a-carboxy-terminal

domain (Poyurovsky et al. 2010) because the

D133p53 isoforms have lost the MDM2-bind-

ing domain present in the amino-terminal end

of p53a, p53b, and p53g.

In addition to proteasome degradation,

D133p53ais also degraded by autophagy dur-

ing replicative senescence. Pharmacological

inhibition of autophagy by baﬁlomycin A1

restores D133p53aexpression levels. The auto-

phagic degradation of D133p53ainduces senes-

cence and is inhibited by direct interaction

of the chaperone-associated E3 ubiquitin ligase

STUB1/CHIP with D133p53a(Horikawa et al.

2014).

Altogether, expression of each p53 isoform

is tightly and differentially regulated, enabling

dynamic and accurate triggering of the appro-

priate cellular response to damage. The diverse

regulations of p53 isoforms expression could be

used as therapeutic targets to trigger controlled

cell-fate outcome.

p53 ISOFORM BIOLOGICAL ACTIVITIES

p53a, p53b(Mp53AS), p53g,D40p53a

(MD41p53a), and D133p53a(ZD113p53a

and MD122p53) have been shown to differen-

tially regulate gene expression and to be bio-

chemically and biologically active either alone

or in combination. D133p53bhas recently been

shown to regulate cell stemness; however, its

molecular mechanism is still unknown (Arsic

et al. 2015).

Over the past 10 years, using diverse human

cell lines and animal models, all data have con-

sistently indicated that the balance of expression

among the p53 isoforms deﬁne the p53-mediat-

ed cell response to trigger after damage, virus/

bacterial infection, or cell signals (Maier et al.

2004; Chen et al. 2009; Fujita et al. 2009; Me-

drano et al. 2009; Davidson et al. 2010; Pehar

et al. 2010, 2014; Ungewitter and Scrable 2010;

Aoubala et al. 2011; Hinault et al. 2011; Slatter

From the TP53 Gene to p53 Protein Isoforms

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et al. 2011, 2015; Terrier et al. 2012; Wei et al.

2012; Bernard et al. 2013; Dichtel-Danjoy et al.

2013; Gambino et al. 2013; Mondal et al. 2013;

Silden et al. 2013; Horikawa et al. 2014; Marcel

et al. 2014; Takahashi et al. 2014; Arsic et al. 2015;

Gong et al. 2015). The p53 isoforms thus play

primordial roles in cell-cycle progression, pro-

grammed cell death, senescence, inﬂammation,

stem-cell renewal and differentiation, aging,

neurodegeneration, glucose metabolism, angio-

genesis, embryo development, and cancer.

The manipulation of p53 isoforms using

splicing-factor inhibitors, autophagy inhibi-

tors, DNA damage, p53 isoform overexpression,

and/or siRNA targeting, speciﬁcally the p53

isoforms, enables the triggering of different

cell-fate outcomes in response to the same dam-

age (Aoubala et al. 2011; Horikawa et al. 2014;

Marcel et al. 2014; Gong et al. 2015). Mechanis-

tically, p53 isoforms oligomerize so that, for

example, the oligomers composed of p53band

p53aregulate different p53-responsive genes

than the ones composed of p53gand p53aor

D133p53aand p53a(Fujita et al. 2009; Aouba

et al. 2011; Bernard et al. 2013; Marcel et al.

2014; Solomon et al. 2014). p53 isoform expres-

sion is cell-type-speciﬁc and several p53 iso-

forms are always concomitantly coexpressed or

are mutually exclusive (Fig. 5). Hence, the p53-

mediated cell response would be the sum of the

activities of each oligomer of the coexpressed

p53 isoforms in a given tissue. It implies that

none of the p53 isoforms, including canonical

p53a, is able to aboli sh the activity or expression

of the other coexpressed p53 isoforms. It is thus

impossible to deﬁne a p53 isoform as oncogene

or tumor suppressor because its activity de-

pends on the cell context. Hence, although

p53bin T cells or normal human ﬁbroblasts

prevents cell growth and induces senescence

(Fujita et al. 2009; Mondal et al. 2013), Marcel

et al. (2014) showed that endogenous p53b/g

in MCF-7 cells inhibit cell proliferation by pro-

moting G

-cell-cycle arrest and cell death under

standard culture conditions, but promote cell

proliferation after treatment with TG003. In

the D133p53-like mouse model (MsD122p53),

although D133p53a/D122p53 inhibits WT p53

tumor suppressor activity, it has recently been

reported that D122p53 proteins enhance the tu-

mor-suppressor activities of an attenuated p53

mutant deleted of the proline domain (MDpro)

(Slatter et al. 2015). This is consistent with clin-

ical studies. Mutant TP53 serous ovarian cancer

patients expressing D133p53ahave better dis-

ease-free and overall survival than mutant TP53

serous ovarian cancer patients not expressing

D133p53a(Hofstetter et al. 2011, 2012; Cham-

bers and Martinez 2012).

CONCLUSION AND INSIGHTS

Based on the data gathered over the past 10 years

by several laboratories using diverse human

cell lines and animal models, we can now assert

that p53 isoforms are physiologically active and

potent proteins. However, their activities are

cell-type-dependent. The misregulation of p53

isoform expression leads to cancer, premature

aging, (neuro)degenerative diseases, inﬂamma-

tion, and embryo malformations. The data

leads us then to realize that a p53-mediated

cell response is not driven by a single protein,

canonical p53, but is in fact the sum of the

activities of the coexpressed p53 isoforms in a

given tissue. Furthermore, a p53-mediated cell

response is broader than just preventing cancer

formation by controlling cell proliferation and

cell death. In fact, the effects of p53 isoform

expression misregulation are consistent with a

physiological role of p53 isoforms in organ

maintenance/regeneration as observed in Dro-

sophila, zebraﬁsh, and mouse p53 isoform ani-

mal models (Jassim et al. 2003; Chen et al. 2005,

2009; Medrano et al. 2009; Davidson et al. 2010;

Dichtel-Danjoy et al. 2013; Hamard et al. 2013;

Simeonova et al. 2013; De la Cova et al. 2014;

Kashio et al. 2014; Senturk et al. 2014; Simo

´n

et al. 2014; Gong et al. 2015). The balance be-

tween the different p53 isoform is crucial in

predicting cell-fate outcome. Therefore, deci-

phering the p53 isoforms combinatorics (p53

isoforms’ code) offers fascinating and impor-

tant new perspectives to treat cancer, degener-

ative diseases and aging.

Cracking the p53 isoforms’ code may seem a

daunting task because of the high number of p53

isoforms and posttranslational modiﬁcations.

S.M. Joruiz and J.-C. Bourdon

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p53 isoforms

p53α

p53γ

p53β

Δ133p53α

Δ40p53α

p53α

p53γ

p53β

Δ133p53α

Δ40p53αp53α

p53α

p53αp53γ

p53γ

p53β

Δ133p53α

Δ133p53β

Δ133p53α

Δ40p53α

Δ133p53α

Δ40p53α

Senescence

Depletion of

Δ133p53α, Δ133p53β

Depletion of

p53β, p53γ,

Δ133p53β

siSplice

si133

Cell death

Differentiation

Senescence

Cell repair

and proliferation

Cell repair

and proliferation

Cell death

Damage

Cell repair

and proliferation

(1)

(2)

(3)

Figure 5. Biological model. (A) p53 isoforms deﬁne cell-fate outcomes in response to intracellular and extra-

cellular cell signals—cell survival/proliferation, senescence, differentiation, or programmed cell death. (B) The

cell type (epigenetic) deﬁnes p53 isoform expression and the possibilities of cell-fate outcomes. (C) In response

to damage, p53 isoforms are differentially regulated and deﬁne cell-fate outcome. (1) In this theoretical cell, the

coexpressed p53 isoforms orchestrate cell repair and allow cell proliferation in response to damage. However, it is

important to note that the cell response to the same damage can be drastically changed by manipulating

expression of only a subset of p53 isoforms using p53 isoform-speciﬁc small interfering RNA (siRNA). (2)

Hence, after depletion of p53b, p53g, and D133p53busing the siRNA siSplice, the cell response to the same

damage is to induce cell death, (3) although, after depletion of D133p53aand D133p53busing the siRNA si133,

the cell response to the same damage is to induce senescence. (This model is based on data in Wu et al. 1994,

Almog et al. 2000, Yin et al. 2002, Chen et al. 2009, Fujita et al. 2009, Ungewitter and Scrable 2010, Aoubala et al.

2011, Terrier et al. 2012, Marcel et al. 2014, Slatter et al. 2015, and Gong et al. 2015.)

From the TP53 Gene to p53 Protein Isoforms

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However, the p53 isoforms are rarely coex-

pressed all together at once; some isoforms are

mutually exclusive, whereas others are always

coexpressed so that the number of p53 isoform

combinations is limited. Cracking the p53

isoforms’ code seems thus an achievable and

necessary task to accurately predict response

to cancer treatment and to control stem-cell re-

newal and differentiation in the treatment of

degenerative diseases and aging. It offers new

exciting perspectives in regenerative medicine.

ACKNOWLEDGMENTS

J-C.B. is a fellow of Breast Cancer Now (Grant

No. 2012MaySF127). All of the p53 antibodies

described in this review (except SAPU) are com-

mercially available or can be requested from Dr.

Borek Vojtesek (vojtesek@mou.cz). The SAPU

antibody can be requested from J.-C.B.

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January 22, 2016 2016; doi: 10.1101/cshperspect.a026039 originally published onlineCold Spring Harb Perspect Med

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Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At the molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here, we report that Banp is indispensable for the DNA damage response and chromosome segregation during mitosis. Zebrafish banp mutants show mitotic cell accumulation and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in banp mutants, suggesting that Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation, chromosome segregation was not smoothly processed from prometaphase to anaphase in banp morphants, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, a DNA replication fork regulator, wrnip1, and two chromosome segregation regulators, cenpt and ncapg, are included in this list. Thus, Banp directly regulates transcription of wrnip1 for recovery from DNA replication stress, and cenpt and ncapg for chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis.

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The term moonlighting proteins refers to those proteins that present alternative functions performed by a single polypeptide chain acquired throughout evolution (called canonical and moonlighting, respectively). Over 78% of moonlighting proteins are involved in human diseases, 48% are targeted by current drugs, and over 25% of them are involved in the virulence of pathogenic microorganisms. These facts encouraged us to study the link between the functions of moonlighting proteins and disease. We found a large number of moonlighting functions activated by pathological conditions that are highly involved in disease development and progression. The factors that activate some moonlighting functions take place only in pathological conditions, such as specific cellular translocations or changes in protein structure. Some moonlighting functions are involved in disease promotion while others are involved in curbing it. The disease-impairing moonlighting functions attempt to restore the homeostasis, or to reduce the damage linked to the imbalance caused by the disease. The disease-promoting moonlighting functions primarily involve the immune system, mesenchyme cross-talk, or excessive tissue proliferation. We often find moonlighting functions linked to the canonical function in a pathological context. Moonlighting functions are especially coordinated in inflammation and cancer. Wound healing and epithelial to mesenchymal transition are very representative. They involve multiple moonlighting proteins with a different role in each phase of the process, contributing to the current-phase phenotype or promoting a phase switch, mitigating the damage or intensifying the remodeling. All of this implies a new level of complexity in the study of pathology genesis, progression, and treatment. The specific protein function involved in a patient’s progress or that is affected by a drug must be elucidated for the correct treatment of diseases.

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Allosteric regulation of protein activity pervades biology as the "second secret of life." We have been examining the allosteric regulation and mutant reactivation of the tumor suppressor protein p53. We have found that generalizing the definition of allosteric effector to include entire proteins and expanding the meaning of binding site to include the interface of a transcription factor with its DNA to be useful in understanding the modulation of protein activity. Here, we cast the variable regions of p53 isoforms as allosteric regulators of p53 interactions with its consensus DNA. We implemented molecular dynamics simulations and our lab's new techniques of molecular dynamics (MD) sectors and MD-Markov state models to investigate the effects of nine naturally occurring splice variant isoforms of p53. We find that all of the isoforms differ from wild type in their dynamic properties and how they interact with the DNA. We consider the implications of these findings on allostery and cancer treatment.

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INT J MOL SCI

A mouse model has often been used in studies of p53 gene expression. Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA’s structure and its interactions with proteins in the translation process. In particular, the 5′-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5′-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5′-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5′-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.

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Chapter

Aug 2022

In this chapter, we review mechanisms by which synonymous codon variations influence gene expression at the mRNA or protein levels. These changes may alter the structure and function of the encoded proteins, leading to diseases, altered disease phenotype, or interfering with protein-drug interactions. In order to appreciate the significance of synonymous mutations, we first look at the concept of codon redundancy and the steps of protein biogenesis which can be influenced by synonymous codon variations.

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Full-text available

Jun 2015

Growing evidence suggests the Δ133p53α isoform may function as an oncogene. It is overexpressed in many tumors, stimulates pathways involved in tumor progression, and inhibits some activities of wild-type p53, including transactivation and apoptosis. We hypothesized that Δ133p53α would have an even more profound effect on p53 variants with weaker tumor-suppressor capability. We tested this using a mouse model heterozygous for a Δ133p53α-like isoform (Δ122p53) and a p53 mutant with weak tumor-suppressor function (mΔpro). The Δ122p53/mΔpro mice showed a unique survival curve with a wide range of survival times (92-495 days) which was much greater than mΔpro/- mice (range 120-250 days) and mice heterozygous for the Δ122p53 and p53 null alleles (Δ122p53/-, range 78-150 days), suggesting Δ122p53 increased the tumor-suppressor activity of mΔpro. Moreover, some of the mice that survived longest only developed benign tumors. In vitro analyses to investigate why some Δ122p53/mΔpro mice were protected from aggressive tumors revealed that Δ122p53 stabilized mΔpro and prolonged the response to DNA damage. Similar effects of Δ122p53 and Δ133p53α were observed on wild-type of full-length p53, but these did not result in improved biological responses. The data suggest that Δ122p53 (and Δ133p53α) could offer some protection against tumors by enhancing the p53 response to stress.

The p53 Isoform Δ133p53β Promotes Cancer Stem Cell Potential

Article

Full-text available

Mar 2015

Cancer stem cells (CSC) are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53β, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53β stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53β expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53β in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53β supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53β isoform. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage

Article

Full-text available

Feb 2015

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.Cell Research advance online publication 20 February 2015; doi:10.1038/cr.2015.22.

Alpha-Enolase Is Upregulated on the Cell Surface and Responds to Plasminogen Activation in Mice Expressing a ∆133p53α Mimic

Article

Full-text available

Feb 2015
PLOS ONE

The p53 protein is a master regulator of the stress response. It acts as a tumor suppressor by inducing transcriptional activation of p53 target genes, with roles in apoptosis, cell cycle arrest and metabolism. The discovery of at least 12 isoforms of p53, some of which have tumor-promoting properties, has opened new avenues of research. Our previous work studied tumor phenotypes in four mouse models with different p53 backgrounds: wild-type p53, p53 null, mutant p53 lacking the proline domain (mΔpro), and a mimic for the human Δ133p53α p53 isoform (Δ122p53). To identify the major proteins affected by p53 function early in the response to DNA damage, the current study investigated the entire proteome of bone marrow, thymus, and lung in the four p53 models. Protein extracts from untreated controls and those treated with amsacrine were analyzed using two-dimensional fluorescence difference gel electrophoresis. In the bone marrow, reactive proteins were universally decreased by wild-type p53, including α-enolase. Further analysis of α-enolase in the p53 models revealed that it was instead increased in Δ122p53 hematopoietic and tumor cell cytosol and on the cell surface. Alpha-enolase on the surface of Δ122p53 cells acted as a plasminogen receptor, with tumor necrosis factor alpha induced upon plasminogen stimulation. Taken together, these data identified new proteins associated with p53 function. One of these proteins, α-enolase, is regulated differently by wild-type p53 and Δ122p53 cells, with reduced abundance as part of a wild-type p53 response and increased abundance with Δ122p53 function. Increased cell surface α-enolase on Δ122p53 cells provides a possible explanation for the model's pro-inflammatory features and suggests that p53 isoforms may direct an inflammatory response by increasing the amount of α-enolase on the cell surface.

A naturally occurring 4-bp deletion in the intron 4 of p53 creates a spectrum of novel p53 isoforms with anti-apoptosis function

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Full-text available

Dec 2014

p53 functions as a tumor suppressor by transcriptionally regulating the expression of genes involved in controlling cell proliferation or apoptosis. p53 and its isoform Δ133p53/Δ113p53 form a negative regulation loop in that p53 activates the expression of Δ133p53/Δ113p53 while Δ133p53/Δ113p53 specifically antagonizes p53 apoptotic activity. This pathway is especially important to safeguard the process of embryogenesis because sudden activation of p53 by DNA damage signals or developmental stress is detrimental to a developing embryo. Here we report the identification of five novel p53 isoforms. p53β is generated due to alternative splicing of the intron 8 of p53 while the other four, namely, TA2p53, TA3p53, TA4p53 and TA5p53, result from the combination of alternative splicing of intron 1 (within intron 4 of the p53 gene) of the Δ113p53 gene and a naturally occurring CATT 4 bp deletion within the alternative splicing product in zebrafish. The CATT 4 bp deletion creates four translation start codons which are in-frame to the open reading frame of Δ113p53. We also show that TAp53 shares the same promoter with Δ113p53 and functions to antagonize p53 apoptotic activity. The identification of Δ113p53/TA2/3/4/5p53 reveals a pro-survival mechanism which operates robustly during embryogenesis in response to the DNA-damage condition. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The p53 control of apoptosis and proliferation: Lessons from Drosophila

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Full-text available

Sep 2014

The canonical role of p53 in preserving genome integrity and limiting carcinogenesis has been well established. In the presence of acute DNA-damage, oncogene deregulation and other forms of cellular stress, p53 orchestrates a myriad of pleiotropic processes to repair cellular damages and maintain homeostasis. Beside these well-studied functions of p53, recent studies in Drosophila have unraveled intriguing roles of Dmp53 in promoting cell division in apoptosis-induced proliferation, enhancing fitness and proliferation of the winner cell in cell competition and coordinating growth at the organ and organismal level in the presence of stress. In this review, we describe these new functions of Dmp53 and discuss their relevance in the context of carcinogenesis.

Modulation of alternative splicing contributes to cancer development: Focusing on p53 isoforms, p53β and p53γ

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Full-text available

Sep 2014
CELL DEATH DIFFER

Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.

Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

Article

Jan 2013

Wrap53, a Natural p53 Antisense Transcript Required for p53 Induction upon DNA Damage

Article

Dec 2016
MOL CELL

Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence

Article

Aug 2014

Δ133p53α, a p53 isoform that can inhibit full-length p53, is downregulated at replicative senescence in a manner independent of mRNA regulation and proteasome-mediated degradation. Here we demonstrate that, unlike full-length p53, Δ133p53α is degraded by autophagy during replicative senescence. Pharmacological inhibition of autophagy restores Δ133p53α expression levels in replicatively senescent fibroblasts, without affecting full-length p53. The siRNA-mediated knockdown of pro-autophagic proteins (ATG5, ATG7 and Beclin-1) also restores Δ133p53α expression. The chaperone-associated E3 ubiquitin ligase STUB1, which is known to regulate autophagy, interacts with Δ133p53α and is downregulated at replicative senescence. The siRNA knockdown of STUB1 in proliferating, early-passage fibroblasts induces the autophagic degradation of Δ133p53α and thereby induces senescence. Upon replicative senescence or STUB1 knockdown, Δ133p53α is recruited to autophagosomes, consistent with its autophagic degradation. This study reveals that STUB1 is an endogenous regulator of Δ133p53α degradation and senescence, and identifies a p53 isoform-specific protein turnover mechanism that orchestrates p53-mediated senescence.

p53 Isoforms: Key Regulators of the Cell Fate Decision

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