HUMAN MUTATION 21:182^191 (2003)

p53 REVIEW ARTICLE

TP53 Family Members and Human Cancers

Jean Bénard,1,2n Setha Douc-Rasy,2 and Jean-Charles Ahomadegbe1,3,4
1
Unité de Génétique Tumorale, Service de Génétique, Département de Biologie Clinique, Institut Gustave Roussy, Villejuif, France; 2Centre
National de la Recherche Scientifique UMR8126, Institut Gustave Roussy, Villejuif, France; 3UPRESS Pharmacologie et Nouveaux
Traitements des Cancers, Institut Gustave Roussy, Villejuif, France; 4Faculté de Pharmacie, UPJV, Amiens, France

For the p53 Special Issue

Based on gene sequence homologies, a p53 (TP53) gene family become apparent with the addition of the
most recently identified p63 (TP73L; formerly TP63) and p73 (TP73) genes to the already known p53.
The p53 gene encodes for a unique protein eliciting well-known tumor suppressor gene (TSG) properties
that mediate cellular response to DNA damage, e.g., cell cycle arrest or apoptosis. In contrast, both
homologues specify an array of isoforms different in their N- and C-terminal domains. Transactivating
isoforms, such as TAp63/p73, show TSG properties similar to p53, while isoforms lacking N-terminal
transactivating domain such as DNp63/p73, induce a functional block against p53 as well as TAp63/p73
activities. Both p63/p73 types of isoforms are involved in development: p63 is critical for epithelial stem
cell renewal and epithelial homeostasis, and p73 is involved in neurogenesis and natural immune
response. These facts support interdependent functions for the p53 family members, which appear linked
together in a complex and tight regulation network to fulfill cellular functions related to DNA damage
and tissue homeostasis maintenance. The lack of p63/p73 mutations in human cancers rule out a typical
TSG role for either of the p53 homologues. Nonetheless, p63 and p73 genes seem strongly involved in
malignancy acquisition and maintenance process because of: 1) their tissue identities, and 2) their close
interplay activities within the p53 family members, and primarily through the negative regulatory role
played by DNp63/p73 isoforms for cell death control and differentiation. Hum Mutat 21:182–191,
2003. r 2003 Wiley-Liss, Inc.

KEY WORDS: p53; TP53; p63; TP73L; p73; TP73; tumor suppressor; splicing; transactivation; apoptosis;
differentiation; DNA damage; development; tumorigenesis; cancers
DATABASES:
TP53 – OMIM: 191170; GenBank: NM_000546 (mRNA)
http://p53.curie.fr/ (p53 Web Site at Institut Curie)
www.iarc.fr/P53/index.html (IARC p53 Mutation Database)
TP73L – OMIM: 603273; GenBank: NM_003722 (mRNA)
TP73 – OMIM: 601990; GenBank: NM_005427 (mRNA)

INTRODUCTION role for p53 homologues in tumorigenesis. Moreover,

The discovery in 1997 of the p73 gene (TP73;
MIM# 601990) at the 1p36 locus, a location known
to be subject to recurrent loss of heterozygosity in
various human cancers [Kaghad et al., 1997], led to
the critical issue of whether this first-identified p53
homologue functions as the archetypal tumor sup-
pressor gene (TSG) p53 (TP53; MIM# 191170). One
year later, the p53 gene family expanded with the
cloning of a second homologue, the p63 gene (TP73L;
MIM# 603273) [Yang et al., 1998], located at 3q27-
ter. This chromosomal region is not subject to loss but
to gain in cancers, and therefore suggests some
oncogenic functions for p63. These apparently para-
doxical findings caused head scratching and skepti-
cism in an oncology research community in search of a
in stark contrast to p53, which is mutated in about
50% of human cancers, it was soon revealed that p73
and p63 genes were not inactivated by mutations in a
large number of cancers from various tissues.
All together, these findings raised a hot question:
Do p53 homologues elicit functions similar to p53?
These issues have been in part elucidated for each p53
homologue from the evidence of an array of isoforms
n
Correspondence to: Jean Be¤nard, UniteŁ de GeŁneŁtique Tumor-
ale, Service de GeŁneŁ tique, Departement de Biologie Clinique,
Institut Gustave Roussy, Villejuif, France.
DOI 10.1002/humu.10172
Published online in Wiley InterScience (www.interscience.wiley.
com).

r2003 WILEY-LISS, INC.

 TP53 FAMILY MEMBERS 183

1 59 142 321 353 397 641

p63α TAD PXXP DBD OD SAM Basic

22% 60% 37%

1 45 113 300 325 363 393

p53 TAD PXXP DBD NLS OD Basic
29% 63% 38%

1 54 131 310 345 390 636

p73α TAD PXXP DBD OD SAM Basic
(A)

P1 TAD DBD OD
1 2 3 4 5 6 7 8 9 10 11 12 13 14
p73α

∆exon2
P2
3bis
∆Np73α
(B)
δ
γ β
ζ
1 2 3 4 5 6 7 8 9 10 11 12 13 14

ε
(C)

FIGURE 1. Comparative gene structures and functional organization of the p53 family members. A: For each gene, the transac-
tivation (TAD), proline-rich (PXXP), DNA binding (DBD), oligomerization (OD), sterile a motif (SAM), and post SAM basic
domains are represented. B: Genomic organization of the p73 gene.The proximal promoter (P1) yields b,c,d,e,f TAp73 isoforms
while the distal promoter (P2) located in intron 3 gives rise to DN isoforms. C: In addition to the a isoform, COOH terminal
splicing leads to b,c,d,e,f p73 isoforms for bothTA and DNp73 molecules species. [Color ¢gure can be viewed in the online issue
at www.interscience.wiley.com.]

expressed in tissues, some harboring transactivation
domain (TAD), a hallmark for transcription factors
with TSG activity, others lacking TAD, e.g., DN-
isoforms, with antagonistic biological properties. There-
fore, unlike their namesake, which stands as a canonical
TSG, each p53 homologue can play either a TSG or an
oncogenic role with respect to its specific isoforms.
In this review we will first present what is currently
known about the two genes and their role during
developmental and adult states, both at the cell and
tissue levels. Thereafter, we will examine their possible
roles in pre-malignancies and cancers.
COMPARATIVE ORGANIZATION AND
TRANSCRIPTION REGULATION IN THE TP53
FAMILY
Akin to their p53 leader, p63 and p73 exhibit the
three typical domains of a transcription factor across
various species: namely an acidic, amino-terminal
transactivation (TA) domain; a central core DNA-
binding domain (DBD); and a carboxy-terminal
oligomerization domain (OD) [Kaghad et al., 1997].
Both p53 ‘‘siblings’’ exert p53-like activities: They can
bind to p53 DNA consensus target sites, transactivate
p53 responsive genes, and induce cell growth arrest or
apoptosis [Kaghad et al., 1997; Jost et al., 1997; Yang
and McKeon, 2000]. The 20kb p53 is organized in 11
exons, whereas p63 and p73 genes are both over 60 kb
and comprise 15 and 14 exons respectively. Unlike the
unique and alternative, but infrequently used, splicing
of the p53 gene, both p63 and p73 consistently give
rise to an array of multiple protein isoforms due to
differential mRNA splicing [Kaghad et al., 1997; De
Laurenzi et al., 1998; Fillippovich et al., 2001] and to
alternative promoter usage [Yang and McKeon, 2000;
Pozniak et al., 2000]. Another specific feature of the
p63 and p73 C-termini is the occurrence of a sterile
alpha motif domain (SAM), which is involved in
protein–protein interactions.
Figure 1 depicts a comparison of the domain
structure of p53 protein and major isoforms encoded
by human p63 and p73. Historically, the C-terminal
complexity was decrypted before the N-terminal
184 BEŁNARD ET AL.
complexity of the 2 gene loci. Most of alternative
splicing occurs at the 30 end and involves more
specifically exons 10 to 13, hence yielding transcripts
that encode protein isoforms with various C-terminal
structures. So far, among the high number of spliced
p63 and p73 isoforms found at the RNA level, three of
them are consistently found at the protein level,
namely a (full structure of both genes), b (splicing of
exon 13), and g (splicing of exons 10 to 12 for p73 and
15 for p63) [Yang et al., 2002]. In contrast, the p53
gene encodes one major transcript yielding a unique
protein with transcriptional activity.
Another striking finding regarding p63 and p73
gene expression regulation has been the occurrence of
two different promoters, P1 and P2, that yield two
distinct classes of proteins [Yang et al., 1998, 2000].
The P1 promoter leads isoforms showing TA domain
with p53 homology. The P2 promoter, located within
intron 3 and over 30-kb downstream, gives rise to N-
terminal-truncated (DN) isoforms with biological
properties opposite to those of p63/p73 TA isoforms,
and lacking TA domain. The (DN) isoforms were
identified first in mouse, then in human [Ishimoto
et al., 2001]. Specific sequences of P1 and P2 bring
about distinct biological properties. E2F-1 binding
sites are present in both promoters, but p53-binding
sites are present only in the P2 promoter [Yang et al.,
2000]. Very recently, two crucial studies have
indicated that: 1) TAp73 directly activates the
transcription of endogenous DNp73 by binding to
the two p73-specific target elements located on P2
[Nakagawa et al., 2002]; and 2) p53 induces DNp73
both at the mRNA and protein levels, as a result of a
p53 direct activation of the P2 promoter [Kartasheva
et al., 2002]. DNp73a-isoform readily associates with
TAp73a/b and p53, as assessed by immunoprecipita-
tion assay, and inhibits their transactivation activities.
On the contrary, TAp63 shows only a marginal DN
p73 transcription. Importantly, the negative feedback
regulation of TAp73 and p53 by their DNp73 target
provides for a novel autoregulatory system modulating
cell survival and death. At the post-translational level,
DN-isoforms are consistently identified in various
tissues expressing p53-homologues, suggesting that
DN-isoform elicits a protein of higher stability than
the TA isoforms transiently expressed and subject to
rapid degradation.
Yet another remarkable distinctive property of the
p53 homologues concerns their degradation process by
MDM2. The turnover of p53, a short-lived protein, is
regulated by ubiquitination through MDM2 binding,
leading to p53 degradation by proteasome, and
thereby limiting p53 accumulation [Haupt et al.,
1997]. Similar to p53, the p73a and b proteins bind to
MDM2 through their N-terminal but this interaction
leads to transcription and apoptosis inactivation, and
does not result in p73 degradation by proteasome
[Balint et al., 1999; Zeng et al., 1999]. So far, p73
ubiquitination has not been demonstrated, but a p73a
modified by a covalent linking with SUMO-1 (small
ubiquitin-related modifier) was found to be more
rapidly degraded by the proteasome than the un-
modified p73 [Minty et al., 2000]. Unlike p53 and
p73, p63 does not bind to MDM2 [Dohn et al., 2001],
revealing yet another difference between p53 family
members.
Therefore one can distinguish two negative feed-
back loops controlling p53 and TAp73, namely the
mdm2 and DNp73 loops, keeping the cell death
trigger under tight control.
A RANGE OF P63 AND P73 ISOFORMS WITH
DISTINCT BIOLOGICAL ACTIVITIES
The combination of C-terminal diversity and acidic
N-terminal regulation, including two distinct promo-
ters, produces at least six major transcripts and
subsequent protein isoforms (a,b,g) for each gene
[De Laurenzi et al., 1998; Schmale and Bamberger,
1997]: TAp63/p73 p53-like isoforms and DNp63/p73
p53-antagonist isoforms (Fig. 1). This unusual gene
regulation suggests a ‘‘two genes in one’’ concept for
p63/p73 since TA and DN-isoforms act respectively as
tumor suppressors and oncogenes [Stiewe et al., 2002;
Stiewe and Pützer, 2002; Grob et al., 2002].
It is well known that p53 TSG properties arise from
its binding to specific DNA sequences and from
transactivation of target genes that specify cell cycle
control proteins (p21, MDM2, GADD 45, 14-3-3-s,
BTG2) and apoptosis (PIG3, Bax). Active p53 was
also shown to play a key role in B lymphocyte, muscle,
spermatogenesis, and neuronal differentiation [Rotter
et al., 1994; Sidell and Koeffler, 1998] as well as in
retinoic acid-mediated terminal differentiation of
embryonic carcinoma cells [Curtin et al., 2001]. p53
is also involved in keratinocytic differentiation,
although it does not determine this process because,
in p53 knock-out mice, keratinocytic differentiation
may be activated by Ca++ treatment.
Another important difference between p53 family
members concerns their interactions with viral onco-
proteins. Indeed if E6 protein, SV40 large T antigen,
and AdE1B 55 kd protein bind to and inactivate p53
during cell transformation, they do not interact with
either p63 or p73 [Irwin and Kaelin, 2001].
When over-expressed in human cells, TA p63/p73
proteins also bind to p53 DNA target sequences,
transactivate p53-responsive genes, and thereby
induce cell cycle arrest, differentiation, and apoptosis
in a p53-like manner [Kaghad et al., 1997; Jost et al.,
1997; Yang et al., 1998] running through p53 most
characteristic biological effects. However, transcrip-
tional activity fluctuates with p63/p73 splice variants
and must be considered. Indeed, TAp63/p73a iso-
forms display a less active transcriptional activity than
TAp63/p73b isoforms in p53 assays [Yang et al., 1998;
 TP53 FAMILY MEMBERS 185

Morphogenesis,
Stress
? Epithelial
UV ? p63 development/
differentiation

∆Np63

DNA ATM Growth

damage ATR, Casein KII arrest
p53
UV DNA-PK
Apoptosis
P300 p53 mutant
PCAF Adenovirus ∆Np73
IR E4orfE6
ATM Neurogenesis,
Mismatch Neuronal
c-Abl p73
repair development/
Cisplatin DNA-PK differentiation

FIGURE 2. Schematic p53 family members pathways. Besides speci¢c developmental and physiological functions, p63 and
p73 participate to p53 genomic guardian function. Upon genotoxic stresses induced by ultraviolet (UV), g irradiation (IR) or
cisplatin, the two homologues interplay with p53 to achieve growth arrest and apoptosis functions. It is now established that
p53 as well asTAp73 induce a direct activation of DN-p73 creating thus a feedback loop to control negatively these functions.
[Color ¢gure can be viewed in the online issue at www.interscience.wiley.com.]

Schmale and Bamberger, 1997] indicative of a
negative regulatory effect of the a isoforms from the
sterile a motif (SAM) (Fig. 1) [Thanos and Bowie,
1999]. Similarly the COOH-terminal region located
in the post SAM domain can have an auto-inhibitory
effect on the TAp63/p73a transactivation [Ozaki
et al., 1999].
Noteworthy, the above-described biological activa-
tion has been demonstrated using forced expression of
TA isoforms of the p53 homologues, likely to mask
differences between the various p53 -family members.
Not to mention that, so far, no binding of endogenous
p63/p73 to their putative target gene promoters has
been demonstrated in vivo by chromatin precipitation.
Nonetheless, differences may exist between human
p53 family members regarding their potency to induce
activation of p53 target genes. Indeed, if p53 leads to
strong p21 and MDM2 induction, p73 was shown to
induce a major and strong 14-3-3 s up-regulation
[Zhu et al., 1998].
In response to various stresses resulting in DNA
damage, p53 is activated by phosphorylation to
perform its cell-cycle arrest and apoptotic tasks.
Similarly, in response to DNA-damaging agents such
as cis-platin and g-irradiation, p73 up-regulation
results in apoptotic response albeit using a pathway
distinct from that of p53. p73 is phosphorylated by c-
abl, the non-tyrosine kinase receptor, which is itself
phosphorylated by the ATM (ataxia telangiectasia-
mutated) protein [Gong et al., 1999; Shaul, 2000].
Therefore, a p73 repair pathway for DNA damage
exists, which is p53-independent (Fig. 2).
So far regarding p63 response to DNA damage,
important information has been gained from UV-
treated human keratinocytes: TAp63 isoforms are up-
regulated while DNp63 isoforms are dramatically
down-regulated in these p63 expressing cells [Liefer
et al., 2000]. As a matter of fact, the DNp63 down
regulation parallels p53 stabilization, which, in turn,
acts as the apoptotic executor of UV-damaged
keratinocytes. These findings provide evidence for a
full interplay between the two main antagonistic p63
isoforms and p53 for DNA-damage response (Fig. 2).
In p53-deficient SaoS-2 cells, oncogenes such as
E2F1, c-myc, and E1A can induce and activate
endogenous p73 protein expression and drive cells to
apoptosis, pointing to a p73 upstream pathway that is
p53-independent [Zaika et al., 1999]. Importantly, a
very recent work shows that, in response to various
stresses (drug, g-irradiation), p53 requires TAp73, as
well as p63, to activate promoters of apoptotic genes
such as NOXA, PERP, and BAX [Flores et al., 2002],
supporting a crucial role for the two homologues in
p53-mediated apoptotic activity.
To sum up, a large body of evidence favors specific
upstream and downstream target gene regulation for
186 BEŁNARD ET AL.
each p53 family member, and for their respective
isoforms, playing a role in non-redundant functions.
Undoubtedly, RNA interference strategies specifically
designed against these isoforms will provide definitive
clues with regard to biological activities of each p53-
family member.
THE TP53 HOMOLOGUES AS DEVELOPMENT
PROTAGONISTS
The respective deficient mice have provided further
insights concerning the physiological role of p53, p73,
and p63. p53 null-mice develop spontaneous tumors
at high frequency without exhibiting a specific
phenotype, in particular during their development
[Donehower et al., 1992]. If active p53 does not
appear to be involved in physiological apoptosis
occurring throughout embryogenesis, it becomes
crucial for DNA-damage-induced apoptosis [Lowe
et al., 1993]. In stark contrast, both p73 and p63 null-
mice show specific developmental defects but no
spontaneous tumors at all [Yang et al., 1999, 2000].
Studies with p63-null mice have revealed the
occurrence of TA and DN isoforms in the stem cell
compartment of stratified epithelia, thereby also
offering a very useful guide for our understanding of
p53-homologues regulation and functions [Yang et al.,
1998, 1999]. p63 knockout-mice are born alive but
display severe deformations of limbs as well as of
epithelia including skin, breast, urothelia, and prostate
[Mills et al., 1999; Yang et al., 1999]. The p63 –/–
mouse skin does not undergo normal development
because it is lacking stratification and differentiation
markers. These mice also lack mammary glands, hair
follicles, and teeth. Such murine phenotypic disorders
were confirmed by p63 mutations in various human
syndromes involving limb and ectodermal develop-
ment. Indeed p63 mutations have been related to
EEC syndrome, a set of autosomal dominant disorders
(ectrodactyly, ectodermal dysplasia, and cleft lip/
palate), as well as to sporadic split, hand-split foot
malformations [Yang et al., 1999; Celli et al., 1999;
van Bokhoven et al., 2001]. In fact, epithelial loss in
p63-deficient mice reflects the inability of the multi-
layered regenerative epithelia stem cells to undergo
asymmetric division. Indeed, in contrast to the
physiological asymmetrical division (one stem cell
providing one stem cell to repopulate the stem
compartment, and one basal cell for differentiation),
all p63 –/– basal cells undergo terminal differentiation,
leading to a complete depletion of the stem cell
compartment. In mice [Yang et al., 1998] and human
[Faridoni-Laurens et al., 2001] epithelia, p63 is highly
expressed in stem cells.
Neuronal defects have been the first phenotypic
traits observed in p73-deficient mice, including high
intracranial pressure and hippocampus dysgenesis
[Yang et al., 2000]. Importantly, it was shown that
DNp73 isoform is predominantly expressed in murine
developing brain and sympathetic neurons, hence
explaining neuronal defects from lack of DNp73. As a
matter of fact, DNp73 isoform was shown to inhibit
neuronal apoptosis from NGF withdrawal by blocking
the p53 pro-apoptotic function [Pozniak et al., 2000].
These seminal findings designate p73 and DNp73
isoforms as determinants of cellular differentiation and
apoptosis in neuronal tissues. Moreover, in developing
Cajal-Retzius cells of the human neocortex, very
recently, a close association was shown between p73
expression and reelin, a glycoprotein involved in
neuronal migration [Meyer et al., 2002]. Also
interesting in this respect, a recent study indicated
that human cytomegalovirus CMV, which causes
severe neuronal defects in utero, induces dramatic
apoptosis inhibition and thus abnormal nerve cell
survival through accumulation of DNp73a [Allart
et al., 2002]. Besides these neurological and cognitive
impairments, p73 null-mice display severe defects in
pheromone-based social behavior, reproduction, and
infection control. Noteworthy, the complex range of
physiological deficiencies correlates with high p73
levels expressed in corresponding tissues of p73-
proficient mice. These phenotypic traits suggest that
the p73 functions sum up a very basic signaling system
in vertebrates, i.e., both sensor and controller
functions of intra and extra cellular signals, which
are integrated in cell homeostasis [Yang et al., 2002].
P63 AND P73 GENES AND TUMORIGENESIS
Although, in contrast to p53 null-mice, p63/p73
null-mice do not develop any spontaneous tumors, the
complex network and regulation system formed by the
main p53-homologue isoform proteins and p53 itself
must be considered in the context of tumorigenesis.
Two levels of interaction between p53 members can be
distinguished: physical interactions and pathway
regulations.
Human tumor-derived p53 mutants can engage
with different p73 and p63 isoforms in a physical
association. In fact, interaction with mutant p53
impairs, in vitro and in vivo, p73 and p63 sequence-
specific DNA binding, consequently inhibiting their
transcriptional activity [Strano et al., 2002]. More-
over, in cells carrying endogenous p53 mutants, p53
homologues are unable to recruit some of their target
gene promoters [Strano et al., 2002]. Therefore, p63
and p73 may contribute to the biological properties of
specific mutants by promoting tumorigenesis and by
conferring selective survival advantage to cancer cells.
The p53 homologues are also integrated in various
p53-independent pathways conducting the comple-
tion of various functions, in particular apoptosis as
previously stressed. However, and strikingly, in
response to DNA damage, p53-dependent apoptosis
relies upon the functionality of both p63 and p73:
 TP53 FAMILY MEMBERS 187

TABLE 1. Meta-Analysis of Mutation, Loss of Heterozygozity, Imprinting and Methylation of p73 in Human Cancers (after
Stiewe and Pˇtzer [2002])
Tumor Mutation LOH Imprinting/methylation
Bladder cancer 0/23 B: 35/46
Breast cancer 1/145 29/194 B: 8/24
CNS-tumors 1/77
Colorectal cancer 0/125 8/46
Esophageal cancer 0/48 11/39 B: 4/9
S:1/9
Gastric cancer 0/82 12/32 B: 37/53
Head and neck cancers 3/58 16/108 B: 8/8
Hematological malignancies 0/91 B: 4/10
methylated:11/35
Hepatocellular carcinoma 1/70 21/71 B: 7/8
Lung cancer 1/114 32/107 B: 30/36
Melanoma 0/68 3/27
Neuroblastoma 2/302 46/246 B:16/25
Ovarian cancer 0/63 33/141
Prostate cancer 0/133 2/38 B: 27/27
Renal cancer 0/27 B: 8/12
S: 2/12

Total 9/1426 213/1049 B:184/258

S:3/21

LOH, loss of heterozygozity; B, biallelic; S, switch.

When using E1A p53-dependent apoptotic fibroblast
system, the lack of p63 and p73 clearly results in the
cell’s loss of functional p53 necessary to undergo
apoptosis [Flores et al., 2002]. Another line of
evidence favoring the interplay between p53 and its
homologues for tumorigenesis is the fact that DNp73
proteins are effective inhibitors of p53 and TAp73
functions [Stiewe et al., 2002]. Consistent with this
fact, endogenous DNp73 protects cells from p53-
dependent apoptosis. The same phenomenon may be
proposed for DNp63 in squamous cells. These
functions provide a plausible explanation for high
p73 and p63 levels in human cancer cells even in the
absence of p63/p73 mutations.
Although completed soon after the p73 and p63
clonings and without any regulation knowledge of
these novel genes, translational studies in cell lines
and fresh tumors of various cancers support the above
proposed mechanisms. Now let’s consider the devel-
opments of recent research saga. Since p73 gene was
shown to reside at 1p36-3, a locus found to be subject
to recurrent loss of heterozygosity in many cancers
(neuroblastoma, breast, colorectal cancers, melano-
ma) and to belong to the smallest overlapping region
[Kaghad et al., 1997], p73 has been proposed as the
long sought after TSG candidate at 1p36 [Versteeg
et al., 1995]. A thorough search of intragenic
sequence variations in the p73 gene resulted in an
unswerving lack of mutations. In a sampling of more
than 1,400 human tumors including solid (breast,
lung, colorectal, esophageal, hepatocellular, renal,
prostate, melanoma, neuroblastoma, and brain tu-
mors) as well as hematological malignancies, only nine
cases (0.6%) showed missense mutations (Table 1)
[Stiewe and Pützer, 2002]. The rare mutations found
(P405R and P425L) had no significant effect on
p73a/b transcriptional activity and growth suppressing
ability [Naka et al., 2001]. Given that p73 was located
in a region so far described as imprinted, the lack of
mutation in the remaining allele raised the possibility
that the loss of transcribed allele would render tumor
cells totally p73-deficient. Although some studies
pointed out monoallelic expression, particularly in
inflammatory breast cancers [Ahomadegbe et al.,
2000], most showed p73 biallelic expression or allele
switching, which invalidates the latter working
hypothesis. p73 expression seems very low and
variable from tissue to tissue: This fact and the poor
quality of the first antibodies raised against p73
prevented western-blot use and favored RT-PCR
analyses. p73 transcript levels were regularly measured
higher in a significant percentage of cancers than in
normal tissues [Zaika et al., 1999; Stiewe and Pützer,
2002]. However, when polymerase reactions were
carried out using cDNA binding domain as substrate,
the semi-quantitative data obtained did not permit an
answer to the question of whether specific TAp73 or
DNp73 isoform was overexpressed and which one
could play a role in tumorigenesis. p73 up-regulation
in some tumors can result from mitogenic oncogenes
as shown in experimental models [Zaika et al., 2001]
that lead to massive apoptosis. Attempts have been
made to relate cancer’s p73 status and disease
prognosis: Studies of large-size patient groups with
hepatocellular carcinomas [Tannapfel et al., 1999],
colorectal carcinoma [Sun, 2002], and breast cancer
[Dominguez et al., 2001] produced a poor prognosis
statistical trend for tumors with high p73 level.
However, again, no evidence pointed to either isoform
species whose transcript level increased: Was it TA or
188 BEŁNARD ET AL.
DNp73? Besides the physiological transcript DNp73,
an N-terminal splice variant lacking exon 2 could be
also found in tissues. Its role in malignancy of various
tissues is under investigation (Fig. 1) [O’Nions et al.,
2001; Douc-Rasy et al., 2002] (Ahomadegbe and
Bénard, in preparation).
So far, two tumor models highlight a direct role for
p73 and p63 in tumorigenesis, neuroblastoma (NB)
and squamous carcinoma cells. Remarkably NB
constitutes a first tumor model in which p73 status
has been thoroughly studied and it provides a
significant clinical impact [Melino et al., 2002]. In
NB, p53 is not mutated but inactivated by cytoplasmic
retention [Moll et al., 1995], making p73 a good
candidate for NB tumorigenesis, given the recurrent
loss of heterozygosity at the p73 locus in these tumors.
Early research provided evidence for lack of p73
mutations in fresh NB, ruling out this gene for a
typical TSG role [Nakagawara et al., 1999; Zaika et al.,
1999; Barrois et al., 2001]. In fact, neuroblastic
tumors (NTs), occurring in early childhood, display a
wide spectrum of differentiation. Recurrent deletions
involving the p73 locus are frequently observed in
undifferentiated NTs. To address the question of
possible p73 implication in neuroblastic differentia-
tion, this gene-expression status was investigated in a
panel of differentiated and undifferentiated tumors.
Although no mutations were found, p73 transcript
profiles differed between undifferentiated and differ-
entiated tumors. The frequency of the transcripts
lacking exon 2 (species 1–3) appeared to be higher in
undifferentiated than in differentiating and differen-
tiated NTs [Douc-Rasy et al., 2002]. In contrast,
products from the use of an alternate promoter
(DNp73) were present in all NTs. In addition, only
DNp73, but no full-length proteins, were detected by
immunoblot, suggesting a greater stability of N-
truncated isoforms [Douc-Rasy et al., 2002]. To
determine the role of DNp73 in NB, the pattern of
this gene’s in vivo expression has been analyzed and
the prognostic significance of its expression evaluated.
Recent results clearly indicate that DNp73 expression
is linked to a reduced apoptosis in NB tumor tissue.
Expression of this variant in NB patients significantly
correlates with reduced survival and progression-free
survival: It is a predictor of poor outcome and
constitutes a prognostic factor independent of age,
primary tumor site, stage, and MYCN amplification
[Casciano et al., 2002a]. At a mechanistic level, it is
noteworthy that P2 promoter functionality in NB cells
and tumor tissues is, at least in part, regulated by
epigenetic mechanisms [Casciano et al., 2002b].
In direct correlation with its role in epithelia
development and homeostatic maintenance, and
consistent with its locus at 3q27-29, a chromosomal
region submitted to gain, p63 levels significantly
increased in squamous cell carcinoma [Hibi et al.,
2000]. Clearly, it is the DNp63 isoform level that was
found to be significantly increased [Hibi et al., 2000;
Crook et al., 2000; Park et al., 2000]. In agreement
with the DNp63 oncogenic role, premalignant and
malignant lesions of the cervix and endometrium
show the highest p63 level in differentiated squamous
cells [Park et al., 2000]. Additional data regarding p73
involvement in cancers came recently from the biopsy
analysis from 50 patients with head and neck
squamous cell carcinoma (HNSCC). It showed a
down regulation of p73 expression in 30% of the
HNSCC associated with high p63 expression in basal
layers as assessed by immunohistochemistry [Faridoni-
Laurens et al., 2001]. Microdissection of HNS
epithelium and HNSCC, currently in progress, is
likely to reveal a DNp63 overexpression concomittent
to this p73 down regulation.
Another finding of interest is DNp63 isoform
overexpression resulting from p63 amplification in
these tumors. This overexpression mediates a decrease
in phosphorylation levels of b-catenin, which in turn
induces b-catenin nuclear accumulation and activates
b-catenin signaling pathway. So, DNp63 isoforms act
as positive regulators of the b-catenin signaling
pathway, hence providing a basis for their oncogenic
properties [Patturajan et al., 2002]. From all these
studies it emerges that p63, p73, and p53 play a role in
concert in the differentiation and carcinogenesis of
HNS epithelium.
In conclusion, the discovery of the p53 homologues
has defined the p53 family. Clearly, p53 is not a lonely
genome guardian counteracting genotoxic insults: It
operates with the mutual assistance of p73 and p63
within a complex network including distinct but
complementary pathways. Lack of p63/73 mutations
in human cancers definitively rule out a typical TSG
role for either p53 homologues. Nonetheless, it is
strongly suggested that p63 and p73 genes may be
involved in acquisition and maintenance of malig-
nancy given: 1) their tissues identities, as well as 2)
the close interplay activities within the p53-family
members, primarily through the negative regulatory
role played by DNp63/p73 isoforms for cell death
control and differentiation. The state of the p53 field
has increased in complexity tremendously over the
last 5 years. New clues may permit researchers to
accurately typify tumors according to alterations of
their respective p53 and p53 homologue pathways.
Undoubtedly, this p53 family genomic signature in
every tumor will become a prerequisite for designing
new therapeutic approaches for cancers.
ACKNOWLEDGMENTS
The authors thank Dr. Daniel CAPUT (Sanofi
Recherches, Labèges, France) for invaluable discus-
sions about their research and Sanofi Recherches for
financial support. The manuscript was edited by
English Booster Ltd, www.englishbooster.com.

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