[Cancer Research 61, 486-488, January 15, 2001]
© 2001 American Association for Cancer Research
p53 Induction Prevents Accumulation of Aberrant Transcripts in Cancer Cells1
Caroline Moyret-Lalle,
Cyril Duriez,
Joris Van Kerckhove,
Christel Gilbert,
Qing Wang and
Alain Puisieux2
Département d’Oncologie Fondamentale et Appliquée, INSERM Unité 453, Centre Léon Bérard, 69008 Lyon, France
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ABSTRACT
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Loss of fidelity of the splicing process occurs during tumor progression
and can have a deleterious effect on genes like tumor suppressor genes.
It was reported recently that the presence of aberrant transcripts of
the TSG101 gene in breast cancer cells was associated
with the mutation of the p53 tumor suppressor gene. On
the basis of this observation, we have analyzed TSG101
transcript patterns in p53-active and p53-inactive cells. Using several
isogenic cellular models, we demonstrate that the induction of p53 in
cancer cells leads to a significant decrease of aberrant transcripts
levels. This indicates a novel implication of p53 in the regulation of
the splicing process.
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Introduction
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Tumor development is driven by genetic and epigenetic events
leading to the alterations of oncogenes and tumor suppressor genes.
During the last few years, several reports have demonstrated that
carcinogenesis induced a deregulation of the process of alternative
splicing (1)
. Tumor suppressor genes can be affected by
such abnormalities, suggesting that splicing defects may participate in
the malignant transformation. The TSG101 gene was first
identified as a candidate tumor suppressor gene, based on the
observation of intragenic deletions in breast cancers (2)
.
These results were denied by later studies that failed to demonstrate
any genomic rearrangements of the gene (1
, 3)
. However, it
is now clear that TSG101 is a frequent target of splicing
defects. Although aberrant transcripts may be found in normal tissues,
the frequency of TSG101 splicing abnormalities increases
during transformation and are consistently observed in breast, cervix,
prostate, liver, and gastrointestinal cancers compared with adjacent
normal tissues (2
, 4, 5, 6)
. Variant transcripts found in
tumors are generally shorter than the wild-type mRNA. They are
generated by exon skipping and alternate RNA processing events.
Increasing levels of variant transcripts in tumors could reflect a
progressive loss of stringent splice control function or an extended
alternative splicing during tumor progression. Several studies have
reported a correlation between the presence of aberrant transcripts and
tumor grades (1
, 4
, 5
, 7)
. Recently, Turpin et
al. (8)
have observed, in breast cancers, an
association of TSG101 aberrant spliced products to
p53 mutation. Overall, these results suggest that an
increasing relaxation of TSG101 splicing fidelity occurs
during malignant progression. To address the specific question of
whether p53 function can interfere with an alternative splicing
process, we have analyzed the presence of aberrant TSG101
transcripts in different isogenic cell models differing only in
p53 status.
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Materials and Methods
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Cell Lines and Treatments.
Parental cell lines used in these studies were obtained from the
American Culture Collection (Rockville, MD). Breast HBL100 and colon
HCT116 cancer cells were grown in McCoy’s medium (Life Technologies,
Inc.) supplemented with 10% FCS. Breast adenocarcinoma MCF7 and
MDA-MB-231 cells and colon carcinoma EB cells were maintained in DMEM
(Life Technologies, Inc.) containing 10% FCS. Hepatocarcinoma HepG2
and Hep3B cells were maintained in Eagle’s MEM (Life Technologies,
Inc.) containing 10% FCS. Esophagus cancer TE-1 cells were cultured in
RPMI 1640 supplemented with 10% FCS. Parental MCF7, HBL100, HepG2, and
HCT116 cell lines express an endogenous wild-type p53 gene.
MDA-MB-231 and TE-1 cells express a mutant p53, whereas
Hep3B exhibits an homozygous deletion of the gene. Cells were plated at
106 cells/100-mm dish 24 h before radiation
treatment. Cells were exposed to 
-irradiation at 6 Gy (a high energy
X-irradiation, 5 or 10 MV, at 
3 Gy/min).
RNA Isolation and
RT-PCR.3
%Total RNA was extracted using Tri-Reagent (Sigma Chemical Co.).
cDNA was synthesized from 3 µg of total RNA with first-strand cDNA
synthesis kit (Amersham Pharmacia Biotech). The reaction mixture was
incubated at 37°C for 1 h, and cDNA was stored at -70°C.
Nested RT-PCR reactions were carried out using the conditions described
by Li et al. (2)
. P1
(5'-CGGGTGTCGGAGAGCCAGCTCAAGAAA-3') and P2
(5'-CCTCCAGCTGGTATCAGAGAAGTCGT-3') primers were used for the first PCR
round for 30 cycles. Second-round PCR for 25 cycles was performed using
two nested primers, P3 (5'-AGCCAGCTCAAGAAAATGGTGTCCAAG-3') and P4
(5'-TCACTGAGACCGGCAGTCTTTCTTGCTT-3'). The RT-PCR products were analyzed
in 1.5% agarose gel.
cDNA Subcloning and Sequencing.
Aberrant PCR products were cut out and purified with the Concert Rapid
gel extraction system (Life Technologies, Inc.). Purified cDNAs were
cloned in the pGEM-T vector (Promega Corp.) and sequenced using an ABI
PRISM 377 DNA sequencer (Perkin-Elmer). Sequencing was carried out in
both directions with the P3 and P4 primers.
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Results and Discussion
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Presence of the 
154–1054 TSG101 Splice Variant
in Cancer Cells.
To study the presence of TSG101 splice products in relation
with p53 status, we first examined the expression
TSG101 transcripts by nested RT-PCR as described by Li
et al. (2)
in wild-type and mutant
p53 cancer cell lines derived from breast (MCF7, HBL100, and
MDA-MB-231), colon (HCT116), and liver (HepG2 and Hep3B) tumors. Using
primers flanking the TSG101 coding region, the predicted
1192-bp full-length transcript (Fig. 1)
was observed in all cell lines after the first round of PCR
amplification. After the second round of PCR, in addition to the
1145-bp, normal-sized TSG101 transcript, bands of smaller
size were detected in all cell lines (data not shown). An 250-bp
transcript was observed in most cases as a major aberrant RT-PCR
product (Figs. 1
and 2)
. Southern blot analysis of the RT-PCR products, using full-length
TSG101 cDNA as a probe, confirmed the authenticity of this
variant TSG101 splice product (data not shown). Sequence
analysis revealed that this fragment (244 bp) was the most frequent
variant TSG101 form identified previously in tumors
(1
, 9
, 10)
. It corresponds to a deletion of 900
nucleotides (nucleotides 154-1054). The deletion junction contains a
donor site-like sequence within the coding exon 1 and an acceptor
site-like sequence within the coding exon 5 (Fig. 1)
. This deletion
encompasses the segment encoding the coiled-coil domain of the TSG101
protein. The resulting aberrant transcript alters the open reading
frame and introduces several in-frame termination codons.
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Fig. 1. Schematic representation of the 154–1054 deleted
TSG101 transcript. The coding exons of the normal
TSG101 transcript, indicated as E1–E6,
as defined by Li et al. (2)
, are shown in
gray. The locations of primers P1, P2,
P3, and P4 used for nested RT-PCR amplification
of TSG101 are indicated. Deletional breakpoints are
indicated by nucleotide numbers. Boldface highlights
donor site-like and acceptor site-like sequences. The nucleotide
numbers are shown according to the nucleotide sequence of
TSG101 cDNA, GenBank accession number U 82130.
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Fig. 2. RT-PCR analysis of TSG101 transcripts in
response to DNA damage. cDNA amplification of full-length (1145-bp) and
154–1054 truncated (244-bp) transcripts by RT-PCR in three breast
cancer cell lines (MCF7 and HBL100 express an endogenous wtp53, whereas
MDA-MB-231 cells express a mutant p53). Untreated (C) or
irradiated (X; 6 Gy) MCF7, HBL100, and MDA-MB-231 cells
were tested 24 h after irradiation. (Results were highly
reproducible from different irradiation experiments, and all RT-PCR
amplifications were performed in triplicate.)
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Levels of TSG101 Spliced Variants Decrease in Stress-induced p53
Cells.
Wild-type p53 (wtp53) is present at extremely low levels in most cells.
In response to DNA damage, the half-life of p53 is increased, and the
protein accumulates in the nucleus where it can act as a
transcriptional factor (11)
. To determine whether
activation of p53 function could be involved in regulation of
TSG101 splicing, TSG101 product patterns were
studied after X-irradiation, using nested RT-PCR in three breast cancer
cell lines (MCF7, HBL100, and MDA-MB-231). As expected, the aberrant
TSG101 product 154–1054 was amplified alongside the
normal transcript in nonirradiated cells (Fig. 2)
. Twenty-four h after
exposure to X-rays, levels of the truncated transcript significantly
decreased in wtp53 cell lines, whereas it remained high in the mutant
p53 cell line (Fig. 2)
. This observation suggested a p53-dependent
mechanism. To assess the specific role of p53, we performed a detailed
kinetic analysis of TSG101 transcripts in response to
-irradiation in MCF7 and HCT116-derived cell lines, exhibiting an
inactivation of endogenous p53. MCF7/MDD2 cells were generated by
stably transfecting breast adenocarcinoma MCF7 cells with a
dominant-negative p53 miniprotein (12
, 13)
. This
COOH-terminal segment of p53 forms stable oligomers with endogenous
p53, leading to the abrogation of sequence-specific DNA binding.
MCF7/MN1 cells were generated from MCF7 cells by transfection of the
antibiotic resistance gene alone. Endogenous wtp53 was inactivated in
HCT116 colon cancer cells by homologous recombination
(14)
. As it was shown in parental cell lines, exposure to
X-rays led to a decrease of the 154–1054 truncated transcript
levels in p53-active cell lines [MN1 and HCT116 p53(+/+)],
whereas levels of the full-length transcript remained unchanged. In
contrast, levels of the abnormal transcript were unaffected in the
two p53-inactive cell lines [MDD2 and HCT116 p53(-/-);
Fig. 3
].
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Fig. 3. Kinetic analysis of TSG101 transcripts
after X-rays in two cellular models exhibiting experimental
inactivation of p53. cDNA amplification of full-length (1145-bp) and
154–1054 truncated (244-bp) transcripts by RT-PCR in MN1/MCF7 and
MDD2/MCF7 cells for 18–48 h after X-rays (A) and in
HCT116 p53 (+/+) and p53(-/-) cells for
0–24 h after X-rays (B).
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Induction of p53 Alone Is Not Sufficient to Down-Regulate the
154–1054 TSG101 Truncated Transcript.
To further study the regulatory role of p53, we tested the pattern of
TSG101 transcripts in three cellular models displaying an
inducible p53 activity:
(a) An expression plasmid encoding a temperature-sensitive
(ts) version of mutant p53 (murine p53 Val135) under the control of a
constitutive promoter was stably transfected into Hep3B, a human
hepatocarcinoma cell line that contains no endogenous p53 protein. As
described previously, ts-p53 protein is inactive at 39°C but is
transcriptionally active upon shift to the permissive temperature of
32°C (15)
.
(b) The esophageal cancer cell line TE-1 expresses an
endogenous mutant p53 (amino acid 272, methionine to valine) that
functions as a ts-p53 (16
, 17)
.
(c) The third cellular model, EB1, was derived from the
human colon carcinoma cell line EB after stable transfection with a
construct containing the wild-type p53 cDNA under the
control of the metallothionein MT-1 promoter (18)
.
In all models, p53 induction leads to a cell cycle arrest (data
not shown). The 154–1054 truncated transcript was detected in
addition to the normal transcript in the three noninduced cell lines.
Levels of truncated transcripts were not modified after p53 induction
(Fig. 4)
, suggesting that wtp53 expression is necessary but not
sufficient to modulate expression of TSG101 aberrant
transcripts.
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Fig. 4. Kinetic analysis of TSG101 transcripts in a
p53-inducible cellular model. cDNA amplification of full-length
(1145-bp) and 154–1054 truncated (244-bp) transcripts by RT-PCR in
Hep3B-tsp53 cells after shifting to the p53-permissive temperature
(32°C) for 2–24 h is shown. Cells grown at 39°C (nonpermissive
temperature) for 2–24 h were used as controls.
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Our results indicate that stress-activated p53 is able to modulate the
TSG101 splicing process. The underlying mechanism remains to
be elucidated. The lack of change in TSG101 transcripts
pattern after experimental induction of p53 (ts-p53; inducible
expression) leads to two main remarks: (a) this suggests
that p53 effects depend either on specific posttranslational
modifications induced by genotoxic treatments or on stress-induced
coactivators; and (b) this indicates that the variation of
the TSG101 spliced transcript levels is not a secondary
consequence of the p53-dependent cell cycle arrest. Consistently,
-irradiation triggers a similar decrease of aberrant transcript
levels in p21Waf1-proficient and -deficient HCT116 cells
(data not shown). Thus, the activation of this cyclin kinase inhibitor,
which is a major p53 target gene, is not required for the p53-dependent
modulation of splicing process.
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ACKNOWLEDGMENTS
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We thank M. Oren and K. Keyomarsi for MN1 and MDD2 cells, B.
Vogelstein for HCT116 p53 (-/-) and p21 (-/-)
cells, and P. Hainaut for TE-1 cells. We also thank C. Ginestet, F.
Lafay, and C. Malet for performing the ionizing radiation.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by grants from the Comité
Départemental de la Ligue de Lutte contre le Cancer, the
Association pour la Recherche sur le Cancer, and INSERM (U453). 
2 To whom requests for reprints should be
addressed, at INSERM Unité 453, Centre Leon Berard, 28 rue
Laennec, 69008 Lyon, France. Fax: 33-4-78-78-27-20; E-mail: puisieux{at}lyon.fnclcc.fr
3 The abbreviation used is: RT-PCR, reverse
transcription-PCR.
Received 8/ 7/00.
Accepted 12/ 6/00.
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ABSTRACT
Introduction
