Oncogene (1999) 18, 7101 ± 7109
ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00
http://www.stockton-press.co.uk/onc
SIAH-1 inhibits cell growth by altering the mitotic process
H Bruzzoni-Giovanelli1, A Faille1, G Linares-Cruz1, M Nemani2, F Le Deist3, A Germani4,
D Chassoux5, G Millot1, J-P Roperch2, R Amson2, A Telerman2 and F Calvo*,1
Laboratoire de Pharmacologie ExpeÂrimentale et Clinique INSERM EP-9932. Institut de GeÂneÂtique MoleÂculaire. HoÃpital SaintLouis. 27 rue Juliette Dodu. 75010 Paris. France; 2Fondation Jean-Dausset - CEPH, Paris, France; 3INSERM Unite 429 HoÃpital
Necker, Paris, France; 4INSERM Unite 363 HoÃpital Cochin, Paris, France; 5INRA Unite 806 Institut Curie, Paris, France
1
SIAH-1, the human homologue of the drosophila seven in
absentia gene, is a p53-p21Waf-1 inducible gene. We report
that stable transfection with SIAH-1 of the epithelial
breast cancer cell line MCF-7 blocks its growth process.
The transfectants show a redistribution of SIAH-1
protein within the nucleus, more speci®cally to the
nuclear matrix, associated to dramatic changes in cell
morphology and defective mitosis. Multinucleated giant
cells (2 ± 12 nuclei in more than 50% cells) were a most
striking observation associated with tubulin spindle
disorganization and defective cytokinesis. There were
also present at high frequency abortive mitotic ®gures,
DNA bridges and persistance of intercellular bridges and
midbodies, along with an increased expression of p21Waf-1.
These results indicate that the mechanism of growth
arrest induced by SIAH-1 in MCF-7 cells involves
disorganization of the mitotic program, mainly during
nuclei separation and cytokinesis.
Keywords: SIAH-1; mitosis; multinucleated cell; cytokinesis; apoptosis; p21
Introduction
The product of the drosophila seven in absentia (Sina) is
required for speci®cation of R7 photoreceptor cells in
the developing eye (Carthew and Rubin, 1990), and
functions downstream of the tyrosine kinase Sevenless
in the Ras-1/Raf-1 pathway (Carthew et al., 1994; Lai
et al., 1996; Neufeld et al., 1998). The murine genome
contains several closely related sina homologues (Siah
1A-D and Siah 2) (Della et al., 1993). Out of four Siah
1 genes, only Siah 1A and Siah 1B have open reading
frames and encode highly homologous proteins. The
murine Siah 1B gene was found to be induced in p53dependent apoptosis by di€erential cDNA display
(Amson et al., 1996). Using the murine Siah 1B gene
as a probe, its human counterpart (SIAH-1/HUMSIAH), was isolated (Nemani et al., 1996). Sina and
SIAH proteins promote ubiquitination and therefore
induce the proteasome-dependent degradation of
interacting proteins such as Tramtrack in drosophila
(Li et al., 1997; Tang et al., 1997), and DCC in
mammalian cells (Hu et al., 1997). The ubiquitin/
proteasome proteolytic pathway catalyses the rapid
degradation of many rate-limiting enzymes, transcrip-
*Correspondence: F Calvo
Received 15 May 1999; revised 20 August 1999; accepted 26 August
1999
tional regulators and critical regulatory proteins, and
plays a primary role in the degradation of the bulk of
proteins in mammalian cells.
SIAH-1 binds Bag-1 protein and inhibits cell growth
(Matsuzawa et al., 1998). Bag-1 is a widely expressed
protein with an ubiquitin-like domain that binds and
cooperates with Bcl-2 in suppressing cell death. Bag-1
binds also to several steroid hormone receptors, some
tyrosine kinase growth factor receptors and Raf-1
kinase, regulating its kinase activity. Moreover, Bag-1
binds and functions as a modulator of Hsp70/Hsc70
family proteins (Takayama et al., 1997).
We recently showed that SIAH-1 was activated
during apoptosis and tumor suppression in p53dependent and -independent cellular models, and
during physiological apoptosis occurring in the
intestinal epithelium (Nemani et al., 1996). Expression
of SIAH-1 was also induced following transfection of
p21Waf-1, a cyclin-dependent kinase inhibitor, along with
spatial nuclear reorganization and tumor suppression
(Linares-Cruz et al., 1998). To investigate the
functional role of SIAH-1, the SIAH-1-cDNA coding
sequence was transfected into the MCF7 human breast
cancer cell line. These cells have functional estrogen
and progesterone receptors, express wild type p53 gene
and low levels SIAH-1 protein. Stable transfection of
MCF-7 cells with SIAH-1 cDNA induced several
phenotypic changes and inhibition of cell growth.
SIAH-1 was redistributed in the nuclear matrix
compartment along with changes in p21Waf-1 expression. A series of striking morphological alterations
were also observed including multinucleation, abnormal mitotic spindle organization and abortive mitosis.
Results
Transfection of SIAH-1
The human breast cancer cell line MCF-7 was
transfected with either SIAH-1 cDNA or with the
vector alone.
RT ± PCR indicated expression of exogenous SIAH1 mRNA (data non-shown), and two clones were
selected for further experiments (1.4, 1.8). Overexpression of the SIAH-1 protein was demonstrated in the
transfected clones by Western blot analysis (Figure
1a).
Following nuclear matrix preparation (nuclear NaCl
extraction resistant compartment after nucleases
treatment), tissue sections from human small intestine
were examined. In a previous work (Nemani et al.,
1996) we showed by RNA in situ hybridization a
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Figure 1 Expression, cellular localization and growth properties of SIAH-1. (a) Western blot analysis of control (MCF-7 and RSV)
and SIAH-1-transfected cells (1.4 and 1.8), with anti-SIAH-1 and a-actin antibodies. (b) Confocal microscopy analysis of SIAH-1
immunoreactivity in nuclear matrix preparations of small intestine. Epithelial cells at the tip of villi exhibit strong labeling (step=
0.35 mm, 6 superimposed confocal planes). (c and d) SIAH-1 immunoreactivity in nuclear matrix preparations in control RSV cells
(c) and transfected cells (d). (e) Growth kinetics of transfected cells (green-triangles and red-squares) vs control cells (RSV, blue) as
measured by optical density
strong SIAH-1 expression in the cells at the top of the
intestine villi, where cells are di€erentiated and die of
apoptosis. Using the anti- SIAH-1 antibody and the
nuclear matrix preparation in tissue sections from
human small intestine, we found the same pattern in
the protein distribution (Figure 1b).
Nuclear matrix preparations were also used to
analyse SIAH-1 location in control and transfected
cells. A ®ne and punctuated cytoplasmic and nuclear
staining was observed. In the control cells (Figure 1c),
SIAH-1 immunostaining showed a ®ne dime outlining
the remnant nuclear envelope. This dime was
disrupted by some bright spots. Some ring-like
structure was also stained within the nucleus. In the
cytoplasm a ®ne di€use and reticulated network was
labeled. In SIAH-1-transfected cells (Figure 1d)
labeling became very strong with reinforcement at
the periphery of the nuclear envelope. Some big bright
dots within this structure were also labeled (Figure
1c,d).
Cell growth
The transfected cell clones showed a marked reduction
of their growth in culture (Figure 1e). The doubling
time was increased in the two clones (more than 96 h),
while controls had a growth similar to MCF7 parental
cell line (36 h).
Growth reduction seemed to be secondary to
abnormal cell division and increased apoptosis.
E€ectively, one of the most striking features noted in
transfected cells was the altered morphology with a
large number of cells displaying an increased size and a
majority exhibiting multiple nuclei.
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H Bruzzoni-Giovanelli et al
SIAH-1 overexpression induces mitosis abnormalities
In transfected cells we observed an important number
of multinucleated cells (Figure 2a ± c). While the
majority of MCF7-RSV cells (490%) had one
nucleus, more than 50% of transfected cells were
polynucleated (i.e. 25% had two nuclei, 15% three
nuclei, 5% four nuclei and 10% of cells had more than
four nuclei. In some cells the presence of a
micronucleus could be seen with dense staining with
Dapi (Figure 2c,d).
Most giant cells exhibited nuclear division but were
unable to accomplish cytokinesis. In the majority of
these cells vacuoles began to appear in the cytoplasm,
increased and fused forming rigid pseudoalveolar
structures and thus pushing the nucleus at the border
of the cytoplasm (Figure 2c and see also Figure 3d).
Finally a large proportion of cells rounded and
detached from the plastic. An other important subset
of transfected cells showed two attached nuclei with
persistent septal furrow. This population frequently
detached from the plastic. These cells underwent
abnormal mitosis ± see Discussion, with morphological
and biochemical characteristics of programmed cell
death. Consequently the number of ¯oating cells in the
transfected clones was higher than in controls.
When cells were stained with alpha tubulin, a set of
abnormalities could be seen in transfected clones.
These were most evident in anaphase and telophase.
In anaphase, as chromosomes were moving to the
poles, DNA bridges could be observed between the two
daughter nuclei and parallel to the spindle which
persisted after mitosis had ended. This was con®rmed
by confocal microscope analysis (Figure 3a,f). In these
spindle ®gures, DNA could be detected after DAPI
staining (Figure 3e,f). In an important subset of cells,
nuclei separation seemed to be abortive, suggesting
bilobulation (Figures 2a ± d; 3c; and see also 4-d ± f). In
some mitotic ®gures evident abnormal spindle organization could be observed (Figure 3g). Asynchrony of
mitotic ®gures in multinucleated cells was also noticed
since mitosis in a given nucleus occurred without
a€ecting the resting state of the other nuclei (Figure
2a). In some polyploid cells, apoptotic ®gures could be
observed.
To further explore mitosis, g-tubulin staining was
performed. In binucleated cells a pair of centrosomes
was located in close proximity of the septum between
non-separated nuclei (Figure 2d, red dots). The
number of centrosomes in multinucleated cells was
variable and without evident relation with the number
of nuclei.
In an important subset of large sized and almost
always bi- or multinucleated transfected cells, round
shaped structures brightly stained by g-tubulin and
SIAH-1 were detected in close proximity of nuclei
(Figure 2c,e). These structures ranging in size from 1 ±
4 mm, were also detected with alpha tubulin and alpha
Figure 2 a and g-tubulin and SIAH-1 staining of SIAH-1-transfected cels. (a and b) a-tubulin staining (red) of SIAH-1-transfected
clones. Note in (a) nuclear cycle asynchrony within multinucleated cells. Interphasic and mitotic nuclei coexist in the same cell. (c)
SIAH-1 staining of transfected clones shows the presence of round structures (red) close to the non-separated nuclei in
multinucleated cells, and sometimes in contact with nucleic acid containing structures. (a-tubulin is shown in green). (d) and (e)
immunoreactivity to g-tubulin (in red). In (d) note duplicated and non migrating centrosomes next to two post mitotic daughter
nuclei with a persistent furrow. (e) g-tubulin stained structures in close proximity of nuclei of transfected cells. Similar structures
were detected with anti a-tubulin (b) and SIAH-1 (c) antibodies. Note the frequency of non-separated nuclei in an important subset
of transfected cells (a ± d). In all pictures chromatin was stained with dapi (blue).
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H Bruzzoni-Giovanelli et al
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Figure 3 Mitosis abnormalities in SIAH-1-transfected cells. Progression through mitosis of interphasic nuclei was followed by
confocal multi¯uorescence analysis using anti a-tubulin antibodies (red) and DNA staining (a ± d in green, e ± g in blue). During
anaphase, nuclei moving to opposite poles exhibit DNA bridges (a, f) associated with a-tubulin staining (a, e). a-tubulin connections
persist in interphasic nuclei (b ± d and g) forming rigid pseudoalveolar structures within the cytoplasm of multinucleated cells (d).
Some cells display an abnormal spindle organization (g). (a ± d) are all false colors
actin antibodies, and sometimes contained DNA
(Figure 2b,c,e).
p21Waf-1 expression
Because of their role in the control of the cell cycle,
the expression of p53 and p21Waf-1 were analysed p53
protein expression was not modi®ed in these cells
(data non-shown). Conversely, p21Waf-1 protein levels
were increased by Western blot analysis and
immunohistochemistry in SIAH-1 transfected clones
(Figure 4a ± c).
The number of cells expressing p21Waf-1 protein by
immunocytochemistry increased from 11% in MCF-7RSV control cells to 56% in clone 1.4, 35% in clone
1.8 (Figure 4b,c).
p21Waf-1 expression was exclusively nuclear with a
staining pattern involving the whole nucleus. Giant
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Figure 4 p21Waf-1 expression in control and SIAH-1-transfected cells. (a) Western blot analysis of p21Waf-1 expression in control
(MCF-7 and RSV) and SIAH-1-transfected cells (1.4 and 1.8). (b ± g) Immunohistochemical analysis of p21Waf-1, in (b) control
(MCF-7 transfected with the vector alone) and (c ± g) SIAH-1 transfected clones showing overexpression of p21Waf-1 protein (red).
(d ± g) Heterogeneous p21Waf-1 expression within multinucleated transfected cells (1006). Cytoplasmic structures were stained with
DiOC6 (green) and nuclei with Dapi (blue)
mononucleated cells were strongly positive for p21Waf-1.
In polynucleated cells, nuclear staining was variable:
some nuclei were strongly positive while others, even in
close contact or non separated, were negative for
p21Waf-1 expression (Figure 4d ± g). The heterogeneous
nuclear staining with antibodies to p21Waf-1 suggests
that these nuclei were at di€erent biological steps.
Analysis of apoptosis
Propidium iodide staining of both adherent and
¯oating cells, revealed a higher percentage of
apoptosis (evaluated as hypodiploid nuclei) after
SIAH-1 transfection. From 31% in the control these
numbers increased to 53 ± 63% in transfected clones.
Tunel assay, which was performed only on adherent
cells, con®rmed these results since 4% of control cells
and 10 ± 15% transfected cells were strongly stained.
Discussion
The SIAH-1 gene was previously shown to be activated
both by p53 and p21Waf-1 in a variety of cell lines and to
be associated with programmed cell death in these
models (Amson et al., 1996; Linares-Cruz et al., 1998).
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H Bruzzoni-Giovanelli et al
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In human tissues, the expression of SIAH-1 gene was
shown to be induced along the intestinal villi in
association with di€erentiation and apoptosis
(Nemani et al., 1996). The present study was therefore
undertaken to better de®ne the role of SIAH-1
following stable transfection of the epithelial breast
cancer cell line MCF-7. Our results show that SIAH-1
overexpression inhibits cell growth through major
alterations of mitosis and increased apoptosis. These
alterations include defects in centrosome duplication
and mobility, defects in spindle dynamics, abnormal
chromosomal segregation and failure of cytokinesis
leading to multinucleated giant cells. We also show the
nucleo-cytoplasmic localization of the protein and its
association to the nuclear matrix.
Using epitope-tagged protein, cellular localization of
SIAH-1 was reported as predominantly cytoplasmic
(Hu et al., 1997) or nucleic (Matsuzawa et al., 1998). It
was also shown that the ring domain of the protein
was required for the formation of punctuated
structures in the cytoplasm. Using a polyclonal
antibody directed against the N-terminal end of
SIAH-1, we found a punctuated pattern both in the
cytoplasm and nuclear matrix with small and large
speckles in the latter, similar to nuclear bodies formed
by acute PML oncoprotein (Koken et al., 1994). The
staining was more important and di€use in the nucleus
of SIAH-1 transfected cells. Since SIAH-1 protein is a
ring ®nger protein with predicted nuclear localization
signal domain, its association with the nuclear matrix
suggests strong interactions with other nuclear matrixresident proteins and DNA. These interactions could
be responsible for alterations in chromosome segregation and other mitotic alterations observed in SIAH-1
overexpressing cells.
After transfection, dramatic morphological changes
occurred in cells, some of them showing an important
size increase. Other cells had a more conserved
morphology and were able to achieve an apparent
normal mitosis, suggesting that the exogenous gene
expression level is crucial for the phenotype. The
presence of DNA bridges between segregating nuclei
evidenced an abnormal separation which could lead to
an asymmetric distribution of genetic material between
daughter cells. This abnormal repartition of genetic
material may thus provoke important di€erences in
daughter nuclei behavior. In multinucleated cells, an
evident asynchrony in cell cycle phases among di€erent
nuclei could be observed, some nuclei undergoing a
nuclear division while a majority of them having an
interphasic aspect.
These multinucleated cells evidently originated from
a failure of cytokinesis. Interestingly, a genetic screen
carried out for mutations that reduce sina activity
showed that nine genes may be required for normal
sina activity. One of these genes corresponds to Peanut
(pnut), a Drosophila septin gene which behaves as a
dominant enhancer of sina. Flies mutant for Peanut
show defects in cytokinesis and die at the larval-pupal
transition. Peanut mutant brains contain a large
number of polyploid and multinucleated cells with
multipolar mitotic spindles and extra centrosomes
(Carthew et al., 1994; Neufeld and Rubin, 1994).
Some mitotic alterations of SIAH-1 transfected cells
were comparable with those described after polo kinase
gene disruption or citron kinase mutant overexpres-
sion. Polo kinase family regulates centrosome separation in ®ssion yeast, Drosophila and human cells
(Glover et al., 1999). Gene disruption in yeast or
antibodies injection in mammalian cells causes failure
of bipolar spindle formation. Monopolar spindles
nucleated by centrosomes which appear duplicated
but are not separated, can be observed. Drosophila
polo mutants also show polyploid cells in larval brains
along with multipolar spindles during male meiosis
(Carmena et al., 1998). Citron kinase, a target of Rho
protein, has been also implicated in cytokinesis. Citron
localizes in the cleavage furrow and midbodies.
Overexpression of citron kinase-active mutants, cause
abnormal cleavage furrow contraction during cytokinesis, with failure of cell separation resulting in
multinucleated cells (Madaule et al., 1998). These
mutants block cytokinesis without markedly a€ecting
cell viability, cell-cycle progression or nuclear division,
conversely to SIAH-1 transfected cells, where the high
frequency of pairs of non separated nuclei suggests
additive alterations occurring during spindle formation
and nuclei separation. Of the multiple observed cell
alterations following SIAH-1 transfection, the most
striking involved tubulin and tubulin-associated functions. The forces required for the microtubule-based
movements are generated by microtubule associated
motor proteins that include dyneins and kinesins.
Mutants of such proteins induce abnormal spindle
formation and function, including abnormal chromosome segregation (Goodson et al., 1997; Mandelkow
and Hoenger, 1999).
Physiological endomitosis and multinucleation
occur in normal human megakaryocytes by abortive
mitosis related to deregulation of mitotic exit and
involves p21Waf-1 upregulation (Vitrat et al., 1998;
Matsumura et al., 1997). Opposite to thrombopioetininduced polyploidization of leukemic cells or bone
marrow megakaryocytes (Nagata et al., 1997), SIAH1-induced multinucleation in MCF-7 cells was not
synchronized and was not preceded by the normal
centrosome duplication and separation. Centrosome
duplication occurs during the G1/S transition in
somatic cells and is coupled to the nuclear cell
cycle. During prophase, the centrosomes initiate
formation of the spindle poles and they nucleate
microtubule assembly. The migration to opposite
poles induce spindle formation and microtubule
dependent motor function is required for centrosome
migration. Failure to coordinate centrosome duplication and mitosis results in abnormal numbers of
centrosomes and aberrant mitoses, with loss of cell
polarity and defective chromosomal segregation and
function, as observed in cancer cells (Lingle et al.,
1998). Centrosome duplication and centriole separation require cyclin-dependent kinase (Cdk2) and can
be speci®cally driven by Cdk2-cyclin E complexes. In
the same way, p21Waf-1 overexpression blocks both the
duplication of centrosomes and centriole separation
in di€erent models (Hinchcli€e et al., 1999; Lacey et
al., 1999; Matsumoto et al., 1999). In SIAH-1
transfected cells, centrosome anomalies in number
(in multinucleated cells), in migration and in
morphology were found. The staining of SIAH-1
transfected cells with g-tubulin, a unique member of
the tubulin family which plays a role in microtubule
nucleation, revealed the presence of non separated
SIAH-1 in mitosis
H Bruzzoni-Giovanelli et al
centrosomes in post mitotic cells. These duplicated
centrosomes were localized in the concavity formed
by two closely tied nuclei. Of note, in some
interphasic SIAH-1 transfected cells, large structures
homogeneously stained by anti g-tubulin antibody
persisted, containing SIAH-1 protein and sometimes
DNA. Antibodies against other cytoskeleton proteins,
like a-tubulin and a-actin, also stained this structure.
The presence of very large and abnormal centrosomes
has been previously reported in several cancer cells
and linked to p53 gene mutation or deletion, leading
to an increased genetic instability (Lingle et al.,
1998). However, MCF7 cells which have no p53
mutation, showed no changes in its localization or
level of protein expression after SIAH-1 transfection.
Along with morphological and mitotic anomalies we
found increased levels of p21Waf-1 protein in SIAH-1
transfected cells. Interestingly p21Waf-1 is activated by
microtubule damage and is required for microtubule
damage checkpoint in mitosis and G1 phase of the cell
cycle, helping to maintain genetic stability (Mantel et al.,
1999; Niculescu et al., 1998). Despite ecient inhibition
of cyclin E, A and B1 dependent kinase activity, cells
released from an S phase block are not delayed in their
ability to progress through S phase by the presence of
p21Waf-1, Signi®cant numbers of cells released from the
p21Waf-1 activated G2 block, undergo endoreduplication
passing through another S phase before mitosis (Bates et
al., 1998). In other models, cells expressing high levels of
p21Waf-1 apparently progressed normally through S phase
and enter in mitosis (Medema et al., 1998). All these
results suggest that p21Waf-1 overexpression, in some
circumstances, is unable to stop cell cycle progression but
may initiate cell signal con¯icts a€ecting cytokinesis and
leading to polyploid and/or multinucleated cells. Multinucleated and giant cells were also observed following
p21Waf-1 transfection of MCF-7 cells (Sheikh et al., 1995).
Previously we showed that SIAH-1 transcription was
increased following p21Waf-1 transfection in U937
leukemia cells (Linares-Cruz et al., 1998) without
evident mitotic abnormalities. On the other hand
reduced p21Waf-1 expression also resulted in gross nuclear
abnormalities and centriole overduplication (Mantel et
al., 1999). Recently, it was shown that p21Waf-1 disruption
prevented g-irradiated cells for a sustained G2 arrest and
allowed them to escape G2 and enter mitosis (Bunz et al.,
1998). Strikingly, the expression of p21Waf-1 in SIAH-1transfected cells was asymmetric in bi- or multinucleated
cells, suggesting an asynchrony of progression in the cell
cycle (best observed in multinucleated cells where nuclear
divisions were completely asynchronous).
The phenotype described here and the interaction of
SIAH-1 with the nuclear matrix suggest that it may be
involved in pathway linking the nuclear cycle with
centrosome cycle and microtubule organization.
Alterations observed may result from interactions of
SIAH-1 protein with the spindle itself or with motor
proteins and DNA. These interactions could explain
abnormalities in mobility and segregation of genetic
material, evidenced by DNA bridges, micronuclei and
the polymorphism of nucleus aspect in multinucleated
cells. These data suggest that SIAH-1 is a good
candidate to play critical roles in the regulation of
major events occurring during mitosis, namely
chromosome segregation, centrosome functions and
cytokinesis.
Materials and methods
Cell line and transfection experiments
The human breast cancer cell line MCF-7 was obtained from
American Type Culture Collection (Rockville, MD, USA).
The cells were cultured in Dulbecco's modi®ed Eagle's
medium (DMEM) supplemented with 10% fetal calf serum.
The c-DNA encoding SIAH-1 (1197 bp) was inserted into
the Eco-R1 site of the pBK-RSV (Stratagene, France). The
resulting pBK-RSV-SIAH-1 or the control vector were
transfected using lipofectin reagent (GIBCO /BRL, France)
according to the manufacturer's instructions. Forty-eight
hours after transfection, clones were selected in culture
medium containing 800 mg/ml geneticin (G418 GIBCO/
BRL) for 3 weeks.
Cell proliferation assays
Cells were plated at a density of 16104 cells in 35 mm Petri
dishes in medium. Triplicate samples were frozen every day.
Cell number was estimated using the hexosaminidase
detection method (Landegren, 1984). The doubling times
were calculated from the initial exponential phase of the
growth curves. All assays were performed in triplicate and
experiments were repeated at least three times.
RNA isolation and RT ± PCR analysis
Total RNA was extracted with Trizol reagent (GIBCO/BRL).
For RT ± PCR analysis of SIAH-1 transfectants, 4 mg of total
RNA was reversed transcribed using Superscript II
preampli®cation system (Life Technologies, Inc). Exogenous
SIAH-1-mRNA was ampli®ed with T3 and T7 primers.
Protein extraction and Western blot analysis
MCF7 clones transfected with SIAH-1 cDNA or control
construct were washed twice with ice cold PBS, collected and
lysed in bu€er (150 mM NaCl, 50 mM HEPES, 2.5 mM
EGTA, 1 mM DTT, 1 mM EDTA, 0.1% Tween 20, 10 mg/
ml leupeptin, 10 mg/ml aprotinin). Lysates were sonicated and
clari®ed at 13 000 g for 4 min at 48C.
Cell lysates (20 mg) were separated by sodium dodecyl
sulfate polyacrylamide gel electrophoresis and transferred to
nylon membranes followed by Western blot analysis with
anti-SIAH-1 polyclonal antibody, anti-human p21Waf-1 monoclonal antibody, anti-human p53 antibody and a-actin
antibody. Immunodetection was performed using the
enhanced chemiluminescence kit for Western blotting
detection (ECL, Amersham).
Antibodies
Polyclonal antibody against N-terminal peptide of SIAH-1
was raised in chicken and anity puri®ed.
Mouse anti human p21Waf-1 (Ab-1, IgG1) OP64 monoclonal antibody was purchased from Oncogene Research
products (USA). Pab1801 is a mouse anti human p53
monoclonal antibody (Novocastra, UK), that recognizes an
epitope between amino acids 32 and 79 at the N-terminus of
both wild type and mutant human P53.
Antibodies to a-tubulin were obtained from Amersham
(USA), g-tubulin from Sigma (France) and a-actin from
Santa Cruz (USA).
Immuno¯uorescence studies
Transfected cells were grown on glass chamber slides as
monolayers (Labteck, Nunc). After 7 days in culture the
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H Bruzzoni-Giovanelli et al
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slides were washed with PBS and ®xed in methanol (2 min)
or in 4% paraformaldehyde (PFA) for 10 min at room
temperature. Endogenous peroxidases were neutralized by
30 min incubation in 3% hydrogen peroxide in PBS. Non
speci®c protein binding was blocked by incubation in 3%
BSA, 0.1% Saponin in PBS for 2 h. Slides were then
incubated 3 h or overnight with primary antibody diluted
in 0.3% BSA, 0.1% Saponin in PBS. After six washes,
staining was revealed using peroxidase conjugated secondary
antibody for 3 h and tyramide-tetramethylrhodamine
(DuPont/NEN, USA) was then applied as ampli®cation
substrate. Nuclei were counterstained with DAPI.
Immuno¯uorescence analysis was performed using a Zeiss
epi¯uorescence microscope coupled to a cooled three-charged
coupled device (3CCD) camera (Lhesa, France), equipped
with a triple band pass ®lter and high numerical aperture
lenses (406, 1.3 NA).
Nuclear matrix studies
Human small intestine was obtained at surgery and was
frozen through an isopentane gradient in liquid nitrogen,
embedded in OCT and cryostat sections (8 mm thick) were
prepared and stored at 7808C until use.
Cells were grown on tissue culture chambers for 3 days.
They were placed on ice, washed with Tris-bu€er (50 mM
Tris-HCl pH 7.5, 3 mM MgCl2, 0.5 mM PMSF), incubated
10 min in ice cold Tris-bu€er with 0.5% Triton X-100,
0.5 mM CuCl2, then incubated at 378C for 30 min with Tris
bu€er containing DNAse I (50 mg/ml) and RNAse-A (20 mg/
ml) according to Berezney et al. (1996). After washes, cells
were incubated in 2 M NaCl in Tris bu€er for 30 min on ice
and ®xed in 2% PFA in PBS. Experiments were achieved
using classical immunohistochemical techniques. Nuclear
matrix analysis of cells and sections of the small intestine
were performed using either classical epi¯uorescence microscopy or confocal scanning laser microscopy (MRC 600 BIORAD).
Apoptosis studies
Apoptosis analysis were performed using ¯ow cytometry, as
described by Nicoletti et al. (1991).
Brie¯y, 66105 cells (adherent and ¯oating cells) were
washed twice in NaCl 300 mM followed by a very short
hypotonic treatment with 600 ml of 0.1% Sodium Citrate,
0.1% Triton X-100 (Sigma) with 50 mg/ml of propidium
iodide. Red ¯uorescence was measured using a FACStar-plus
¯ow cytometer (Becton Dickinson). Hypodiploid cells were
counted as apoptotic.
Apoptosis was also measured by TUNEL assay (terminal
deoxinucleotidyl transferase mediated UTP end labeling)
(Gavrieli et al., 1992). For in situ analysis, fragmented
DNA ends within apoptotic cells were end labeled with
biotinylated poly dU by terminal deoxinucleotidyltransferase
and revealed using avidin-conjugated peroxydase and
tyramide-¯uorescein isothiocyanate as substrate (BruzzoniGiovanelli et al., 1998).
Acknowledgements
We wish to thank Dr S Gisselbrecht for helpful discussions. H Bruzzoni-Giovanelli is recipient of a grant from
the French Government and was recipient of a grant from
Conicyt Uruguay. This work was supported by grants from
ECOS program and Association pour la Recherche Contre
le Cancer (ARC) France to F Calvo.
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