By
From the * Biochemistry Laboratory, Istituto Dermopatico dell'Immacolata-IRCCS, Department of
Experimental Medicine, University of Rome "Tor Vergata," 00133 Rome, Italy; and the Laboratory of
Gene Expression, Fondazione Andrea Cesalpino, University of Rome "La Sapienza," 00161 Rome, Italy
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Abstract |
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p73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, , or a shorter
mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of
two new p73 splicing variants,
(splicing out exon 11) and
(splicing out exons 11, 12, and 13). Both
and
p73 variants are expressed in human peripheral blood lymphocytes, primary
keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs
in both primary cells of different lineage and established cell lines even within the same type of
tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73
nor p73
interact with p53,
whereas p73
showed strong interactions with all p73 isoforms, and p73
binds efficiently
p73
and p73
but only weakly p73
. At the functional level, p73
is significantly less efficient
in activating transcription of the p21Waf1/Cip1 promoter than p53 or p73
, whereas the effect of
p73
is intermediate and comparable to that of p73
. The ability of the different p73 variants
to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional
activity on the p21Waf1/Cip1 promoter: p73
is the most efficient in inhibiting colony formation, whereas p73
is almost ineffective. Our results suggest that p73 isoforms may be differentially
regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and
growth suppression activities by affecting heterodimer formation.
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Introduction |
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The p53 tumor suppressor gene (1, 2) is the most frequent target for genetic alterations in human cancers (3, 4). Attempts to find p53-related genes by low stringency hybridization or by degenerate PCR techniques, in analogy to the pRb family of tumor suppressors, have repeatedly failed. A new gene, p73, has recently been identified that encodes a protein with structural and functional homology to p53 (5). The biological activities of the p53 protein that have been implicated in tumor suppression include growth arrest at both the G1/S and the G2/M transitions of the cell cycle (2, 6), and induction of apoptosis (7). To exert most if not all of its functions, p53 acts as a transcriptional activator on numerous genes that contain specific DNA-binding sequences on their promoter, such as p21Waf1/Cip1 (8, 9) and gadd45 (10), or as a transcription repressor on genes that do not contain such sequences, probably by sequestering transcription factors (11, 12). Similar to p53, p73 can activate transcription of p21Waf1/Cip1, a gene involved in cell growth arrest, and can induce apoptosis when overexpressed (5, 13). p73 gene maps to the short arm of chromosome 1 in a region (1p36.33) that is frequently deleted in neuroblastomas (14, 15), suggesting that it may play a role in the development of these tumors. However, allelic loss in lung cancers does not show imbalance of expression or mutations of the remaining allele (16), and p73 mutations do not appear to be frequent in prostate carcinoma (17).
Despite its structural and functional similarities to p53,
p73 is not induced by DNA damaging agents (UV irradiation or actinomycin D) known to activate p53, suggesting
that p73 may have different functions and may be involved
in the cellular response to different stimuli (5, 18). p73,
which is capable of interacting with p53 in a yeast two-
hybrid system (5), may also modulate the function of the
latter. The full-length protein, p73, has been shown to be
expressed in parallel with a shorter splice variant, p73
, in
which the splicing of exon 13 results in a much shorter COOH terminus (5). Here we identify and characterize
the functional properties of two new splice variants of the
p73 gene, called p73
and p73
, differentially expressed
both in normal human cells and in tumor cell lines.
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Materials and Methods |
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p73 Cloning.
Reverse transcription (RT)-PCR for all p73 variants used the following primers derived from the published p73 cDNA sequence (sequence data available from EMBL/GenBank/DDBJ under accession no. Y11416): 5'-TTCTGCAGGTGACTCAGGCTG-3' for RT; and 5'-TTAGCGCTATGGCCCAGTCCACCGCC-3' (sense primer containing an in-frame NheI site) and 5'-CGAGGCCTCAGTGGATCTGC-3' (antisense primer) for PCR amplification. cDNA synthesis was performed using 1 µg of total RNA, extracted from the SH-Sy5y cell line using TRIZOL (Life Technologies, Inc., Gaithersburg, MD), in 20 µl of reaction buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, dNTPs 0.5 mM each, primers 1 µM each, and 200 U of SuperScript RNaseHp73 Transcript Analysis.
To detect expression, all four p73 splicing variants were amplified by radioactive RT-PCR using the following primers: 5'-TTCTGCAGGTGACTCAGGCTG-3' for RT; and 5'-ACTTTGAGATCCTGATGAAG-3' (sense primer) and 5'-CAGATGGTCATGCGGTACTG-3' (antisense primer) for PCR amplification. cDNA synthesis was performed as described above, starting with equal RNA concentrations (1 µg). Radioactive PCR reaction was performed in 50 µl of reaction buffer containing 5 µl of the RT product, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl2, dNTPs 0.2 mM each, cold primers 0.4 µM each, plus 40 nM each of [Tissue Culture.
All of the cell lines were grown in a 1:1 mixture of MEM and Ham's F-12 medium supplemented with 10% heat-inactivated FCS, 1.2 g bicarbonate/l, 1% nonessential amino acids, and 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, at 37°C in a humidified atmosphere of 5% CO2 in air. SAOS-2 cells were grown in DMEM supplemented with 10% fetal bovine serum.Lymphocyte Analysis.
PBLs were purified on a Ficoll gradient, labeled by direct immunofluorescence with anti-CD4-PE and anti-CD8-FITC mAbs (Becton Dickinson, San Jose, CA), and sorted by flow cytometry in a FACSCalibur® (Becton Dickinson) according to the manufacturer's protocol. Analysis of the purified population showed a purity of 97%.Yeast Two-hybrid System.
Nucleotide sequences corresponding to amino acids 72-393 of p53, 85-636 of p73Luciferase Assay.
SAOS-2 cells were transfected by the calcium phosphate method with reporter plasmid containing luciferase cDNA under the control of the p21Waf1/Cip1 promoter, together with p53 or p73s expression vectors. 48 h after transfection, cells were lysed and luciferase activity was quantified as described (20).Growth Suppression Assay.
SAOS-2 cells were transfected in 100-mm plates with the indicated pcDNA3-derived vectors using the calcium phosphate method. 48 h later, cells were placed under G418 (GIBCO BRL) selection (700 mg/ml). After 2 wk, cells were fixed, stained with crystal violet, and photographed, as described (21).Western Blot Analysis.
SAOS-2 cells were transfected using the calcium phosphate method and lysed 48 h later in 50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% NP-40, 0.5 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.5 mM PMSF. The lysate was cleared by centrifugation, and 30 µg aliquots of cell extract, as determined by the Bradford method, were resolved by electrophoresis in a 10% SDS-polyacrylamide gel, transferred to a nylon membrane, and probed with an anti-HA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunocomplexes were detected by a chemiluminescence-based system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) according to the manufacturer's instructions. ![]() |
Results and Discussion |
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The p53 tumor suppressor gene encodes for a phosphorylated protein, 393 amino acids long and highly conserved throughout evolution (22). At the structural level, the four functional domains (1, 2, 23) include the transcriptional activation domain (residues 1-42), the DNA-binding domain (residues 102-292), which is the region most frequently mutated in cancer, the oligomerization domain (residues 324-355), and the COOH terminus domain that allosterically regulates the binding of the central region to DNA. The NH2-terminal transcriptional activation domain binds the members of the CBP/p300 family of transcriptional adaptors/coactivators, which are recruited for most p53 biological responses (24). p73 homology to p53 is very high in the transactivation domain (29% identity), in the DNA-binding domain (63%), and in the oligomerization domain (38% [5]).
p73 gene encodes two distinct polypeptides, p73 and
p73
. The latter arises from the alternative splicing of exon
13 in the p73 transcript (5). With the exception of the last
five residues which are unique to the shorter p73
, the rest
of the protein is identical to the corresponding regions in
p73
. To evaluate whether these splicing variants might
be differentially expressed in different tissues or cell lines,
we systematically characterized p73 transcripts using RT-PCR. While amplifying the 3' end of the p73 mRNA
from the SH-Sy5y neuroblastoma cell line, we identified two amplification products of 386 and 154 bp, in addition
to the expected 535- and 440-bp bands, deriving from the
p73
and p73
transcripts, respectively. We cloned and sequenced all cDNAs, and found that they corresponded to
four different splice variants of the gene (Fig. 1 A). We
named the two new forms p73
and p73
. p73
mRNA
was generated by splicing of exon 11, whereas p73
transcript lacked exons 11, 12, and 13. The splicing of exon 11 in p73
resulted in a frame shift from the original reading
frame leading to the translation of 76 amino acids, different
from the original sequence (from amino acid residue 400 to
residue 475), followed by a premature stop codon (Fig. 1
B). This 76 amino acid sequence did not show any significant similarity to other known peptide sequences in the database. The resulting p73
protein is 161 amino acids
shorter than the p73
and 24 amino acids shorter than the
p73
form, bearing a new COOH terminus that is different from both previously described forms. The splicing of
exons 11, 12, and 13 in p73
resulted in a shorter truncated
form of p73 of 403 amino acids (Fig. 1, A and B). p73
bears the most striking resemblance to p53, as it completely
lacks the COOH terminus extension that differentiates the
other forms of p73. However, both p73
and p73
still
contain the regions homologous to the transcription activation domain, the CBP/p300 coactivator binding domain,
and the DNA-binding and oligomerization domains of p53.
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The relative
expression of the p73 isoforms in normal and neoplastic
cells was examined using a radioactive PCR that amplifies exons 10-14 in the p73 mRNA (bp 1198-1735 of the full-length mRNA). This strategy identified four bands of 535 (p73), 440 (p73
), 386 (p73
), and 154 bp (p73
), thus
distinguishing all four splicing variants with a single PCR.
Fig. 2 A shows the results obtained in human PBLs, primary keratinocytes, eight neuroblastoma cell lines, and
seven other tumor cell lines, including glioblastoma, melanoma, hepatoma, and leukemia. Fig. 2 B shows the linearity of the PCR reaction in the same experimental conditions used to amplify p73 and p53. The expression pattern
of the four p73 splicing variants differs both in primary cells
of different lineage and in established cell lines even within
the same type of tumor. Two cell lines, Jurkat lymphoma
and SMS-KCNR neuroblastoma, were completely negative for p73 expression using our highly sensitive radioactive PCR. Interestingly, normal human lymphocytes lacked p73
and expressed only the p73
, p73
, and p73
forms,
whereas normal human keratinocytes from a skin biopsy
expressed p73
, p73
, and p73
but not p73
. When
CD4+ and CD8+ enriched lymphocyte populations (97%
enrichment after flow cytometry cell sorting) were analyzed, we found that the different forms of p73 were selectively expressed (Fig. 2 C). These results indicate that, although a larger panel of normal tissues needs to be
analyzed, the newly identified p73
and p73
forms are indeed expressed in normal primary cells and do not represent abnormal transcripts expressed only in transformed cell
lines. Our findings also suggest that the various p73 isoforms may play distinct roles in cells of different lineage or
at different differentiation stages within the same lineage.
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To study homotypic and heterotypic interactions between the p73 and p73
and both the other p73
isoforms and p53, we used the yeast two-hybrid system
(Fig. 3 A). As reported previously (5), both the
and
forms interact weakly with p53, whereas the
and
forms
show no interaction with p53. The different forms of p73
interact with each other with variable intensity. p73
showed strong interactions with all p73 isoforms, including itself. The interaction with p73
was the strongest. p73
homodimerizes efficiently and binds p73
and p73
. The
interaction of p73
with the
isoform is much weaker
than that of p73
. We also confirmed that p73
has a very
low tendency to form homotypic interactions, whereas
p73
homodimerizes strongly and binds weakly to p73
. Comparable results were obtained using the different p73
isoforms in either bait or prey configuration in the two-
hybrid assay. Altogether, these results indicate that all homotypic and several heterotypic interactions are possible
between the p73 variants, and suggest that the same may
occur in vivo to fine-tune the system.
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p73 and
p73
have been previously shown to share with p53 the
ability to activate the transcription of common target regulatory sequences (13) and to increase the expression of the
p21Waf1/Cip1 gene (5). To assess whether the two new splicing variants of p73 we identified are also transcriptionally
active, we tested their ability to transactivate the p21Waf1/Cip1
promoter-driven luciferase reporter in the osteogenic sarcoma cell line SAOS-2, which harbors a homozygous deletion of the p53 gene and does not synthesize p53. The
results shown in Fig. 3 B demonstrate that p73
is significantly less efficient in activating transcription from the
p21Waf1/Cip1 promoter than p53 or p73
, whereas the effect
of p73
is intermediate and comparable to that of p73
.
The above-described differences are not due to variations
in the protein levels of the exogenously expressed p73 variants, as shown in Fig. 3 C. Coexpression of either p73
or
p73
with p73
results in a decrease of p21Waf1/Cip1 promoter activation compared with p73
alone. Interestingly, p73
, which activates transcription poorly by itself and interacts strongly with p73
, exerts no inhibitory effect. The
molecular basis for these observations is still unclear. In fact,
it remains to be established whether the various p73 homodimers and heterodimers have different DNA-binding
capacity, intrinsic transcription activation activity, or ability
to recruit the transcriptional adaptors/coactivators of the
CBP/p300 family. In keeping with their ability to increase p21Waf1/Cip1 levels in the cell, both p73
and p73
can induce growth arrest when transfected into SK-N-AS (5) or
SAOS-2 cells (13). We then tested the effect of exogenously
expressed p73
and p73
on cell growth in SAOS-2 cells
using a standard colony formation assay. To this aim, cells
were transfected with the expression vectors either for each
p73 isoform or for p53. After G418 selection for 2 wk,
drug-resistant colonies were stained and counted. The ability of the different p73 variants to affect cell growth in p53
null SAOS cells correlates well with their transcriptional activity on the p21Waf1/Cip1 promoter. p73
was the most
efficient in inhibiting colony formation, and p73
was almost ineffective (Fig. 4).
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In conclusion, we have identified and functionally characterized two new p73 splicing variants named p73 and
p73
. Both variants are expressed in human normal cells as
well as in tumor cell lines of different origin. Three lines of
evidence indicate that the p73 system by itself is likely to
provide a complex regulatory mechanism devoted to the
control of cell growth: (a) the finding of different expression patterns of the four p73 splicing variants in normal and
neoplastic cells; (b) the network of homodimeric and heterodimeric interactions of the various p73 isoforms and p53; and (c) the differences in their transcriptional proficiency and cell growth arrest capacity. The selective ability
of the p73 variants to interact with and possibly to modulate the functions of p53 suggests an even more complicated scenario. Indeed, despite the numerous structural and
functional similarities between p73 and p53, we do not yet
know which stimuli regulate the activity of the various p73
isoforms and their relative ratios in the cell. Unlike p53,
which is usually present in small amounts in normal cells
(22, 27) and is induced by DNA-damaging agents and radiation (28), p73 is not upregulated in response to DNA
damage. Although further investigation is necessary to determine how p73 isoforms are activated and/or regulated,
their biological effects may well include cell growth arrest
and possibly the induction of cell death.
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Footnotes |
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Address correspondence to Gerry Melino, Biochemistry Laboratory, IDI-IRCCS, c/o Department of Experimental Medicine (F153/D26), University of Rome "Tor Vergata," via Tor Vergata 135, 00133 Rome, Italy. Phone: 39-6-20427299; Fax: 39-6-20427290; E-mail: gerry.melino{at}uniroma2.it
Received for publication 15 June 1998 and in revised form 11 August 1998.
This work was supported by Fondazione Telethon (grant E413), Ministero della Sanità, Associazione Neuroblastoma, and Associazione Italiana Ricerca sul Cancro (to G. Melino); and by CNR-ACRO Project, Associazione Italiana Ricerca sul Cancro, Fondazione Telethon, EC Biomed-2, and Fondazione Andrea Cesalpino (to M. Levrero).
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