1 Department of Neuroscience, University of Roma, Tor Vergata, Via Montpellier
1, 00133 Roma, Italy
2 Nuclear Reprogramming Laboratory, Division of Gene Expression and Development,
Roslin Institute (Edinburgh), Midlothian, Scotland, EH25 9PS, UK
* Authors for correspondence (e-mail: biocca{at}med.uniroma2.it and prim.singh{at}bbsrc.ac.uk )
Accepted 1 February 2002
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Summary |
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Key words: Intracellular antibodies, scFv fragments, Chromodomain, HP1 proteins, Heterochromatin, Apoptosis
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Introduction |
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The chromodomain motif is highly conserved, which allows the isolation of
HP1 and Pc-like proteins from mammals
(Singh et al., 1991). There
are three HP1 proteins in mammals. In mouse, they are designated HP1
,
M31 (HP1ß) and M32 (HP1
), and in man HP1
, HP1ß and
HP1
. HP1 proteins have a similar structural organization
(Jones et al., 2000
), are
small (around 200 amino acids) and possess a CD at the N-terminus, which is
immediately preceded by a stretch of negatively charged amino acids (usually
glutamic acid). The CD consists of anti-parallel, three-stranded ß-sheets
packed against an
-helix (Ball et
al., 1997
). Towards the C-terminus, HP1 proteins contain a related
sequence, known as the chromo shadow domain (CSD)
(Aasland and Stewart, 1995
). At
the level of 3D structure, CD and CSD show remarkable similarity to the
histone-like archeobacterial proteins Sac7d and Sso7d but lack the surface
charge that is necessary for DNA binding
(Ball et al., 1997
;
Brasher et al., 2000
).
In mammals, the functions of HP1 proteins are wide ranging
(Jones et al., 2000).
HP1ß is a dosage-dependent modifier of a variegating position-effect
(Festenstein et al., 1999
),
like Drosophila HP1. This ability to silence gene activity has been
utilized by mammalian transcriptional repressors: there is good evidence that
HP1 proteins are recruited by several transcriptional repressors, including
the KRAB-ZFPs, which are the largest family of repressors in mammals
(Ryan et al., 1999
). The
mechanism by which HP1 proteins repress gene activity has become clearer from
a known interaction with the histone methyl-transferase Suv(3)9h1
(Aagaard et al., 1999
;
Melcher et al., 2000
). It is
thought that methylation of H3 histone at lysine 9 forms a substrate for
binding of HP1 proteins and repression
(Rea et al., 2000
;
Lachner et al., 2001
;
Bannister et al., 2001
). This
role for HP1 proteins in silencing gene activity is likely to be conserved
from yeast to man (Wang et al.,
2000
).
A role in the recruitment of nuclear envelope (NE) precursors at the end of
mitosis has also been shown (Kourmouli et
al., 2000). HP1ß and HP1
accumulate at the polar
surfaces of both human and murine anaphase chromosomes. This polar
accumulation closely resembles the cap-like structure that develops at the
early stages of NE reassembly (Georgatos
and Theodoropoulos, 1999
). Consistent with a role in nuclear
envelope re-assembly, an N-terminal fragment of HP1ß, which contains a
NE-binding site, acts as a dominant negative and abolishes targeting of
membrane proteins to the surfaces of chromosomes
(Kourmouli et al., 2000
).
Thus, the presence of HP1 proteins at the polar surfaces of anaphase
chromosomes seems to provide a `platform' that is used by NE precursors for
the assembly of a new nucleus. An additional mitotic role for HP1 proteins is
also suggested from the study of INCENPs, where HP1ß is required for
targeting of this protein to the mitotic spindle
(Ainsztein et al., 1998
).
Finally, cytogenetic studies have indicated two roles for HP1 proteins
during mammalian spermatogenesis. The first is in sex chromosome inactivation
(Motzkus et al., 1999;
Hoyer-Fender et al., 2000
).
The second concerns a role in chromosome cohesion, which is required for
appropriate segregation of the sex chromosomes during male meiosis
(Turner et al., 2001
).
In order to determine the importance of HP1 chromodomain proteins for cell
survival, we have used the intracellular antibody approach (for reviews, see
Biocca and Cattaneo, 1995;
Cattaneo and Biocca, 1997
).
Intracellular antibodies, in particular single-chain Fv (scFv) fragments, have
been successfully expressed inside cells to ablate the function of oncogene
products (Biocca et al., 1993
),
HIV viral proteins (Mhashilkar et al.,
1995
; Levy-Mintz et al.,
1996
) and demonstrated to induce viral resistance in plants
(Tavladoraki et al., 1993
).
The functional basis of intracellular antibodies is closely linked to their
ability to interact with their target antigens in vivo. This interaction
allows either a direct neutralizing effect or the dislodgment of the antigen
from its normal intracellular location which, by this mechanism, inactivates
its function (Lener et al.,
2000
; Cardinale et al.,
2001
).
In this paper we report the cloning and intracellular expression of two scFv fragments directed against non-overlapping epitopes of the HP1 CD motif. Different constructs have been designed to express the anti-CD scFv fragments in the nucleus and in the cytoplasm of mammalian cells. The intracellularly expressed scFv fragments are shown to interact with and dislodge the HP1 protein(s) from their heterochromatin localization in vivo. In vivo interaction and inhibition of the endogenous HP1 CD proteins leads to a cell-lethal phenotype. Cells undergo an irreversible p53-independent cell death program, suggesting that there is an absolute requirement for HP1 chromodomain function in mammalian cells.
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Materials and Methods |
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Expression of scFvs at the surface of the filamentous phase M13 and
phage ELISA
pDAN-transformed colonies of E. coli DH5F' bacteria
were infected with the helper phage M13K07 (Amersham Pharmacia) to a final
concentration of 1010 pfu/ml for 30 minutes at 37°C, and, after
centrifugation, the bacteria were grown overnight at 30°C in fresh LB,
containing 100 µg/ml amplicillin and 25 µg/ml kanamycin. Supernatants,
containing scFv phages, were analyzed for binding to coated HP1ß-GST in
phage-enzyme-linked immunosorbent assay (ELISA)
(Roovers et al., 1998
).
Positive clones were further analyzed by fingerprinting and sequencing.
scFv purification
Anti-lysozyme scFv D1.3 (Hawkins et
al., 1993) (kindly provided by G. Winter) and selected
anti-chromodomain scFv fragments were expressed in E. coli HB2151
non-suppressor strain. 11 of transformed bacteria was induced by 1 mM
isopropylthiogalactoside for 5 hours at 37°C, and the extracts were
obtained by lysis on ice for 30 minutes in 20 ml buffer (8 M urea, 0,1 M
NaH2PO4; 0,01 M Tris-Cl, pH 8 containing 1 mg/ml
lysozyme) and sonication. The scFvs were purified by affinity chromatography
using Ni-NTA agarose (Qiagen) and analyzed by SDS/PAGE followed by western
blotting using the mouse anti-tag monoclonal antibody SV5 (Invitrogen).
ELISA assays
Purification of recombinant HP1, HP1ß, HP1
, mPc1 (M33)
and CD expressed as fusion proteins with GST was performed as described
previously (Smith and Johnson,
1988
). The ELISA assay was carried out by coating the plate with a
solution of 10 µg/ml GST fusion proteins in PBS and a solution of 3mg/ml of
lysozyme (Sigma). After saturation with 2% w/v BSA/PBS solution, 10 µg/ml
scFv fragment solution (diluted in PBST 2% BSA) was added. Detection was
performed using mouse anti-SV5-tag monoclonal antibodies (Invitrogen) and goat
anti-mouse linked horseradish peroxidase (Amersham Pharmacia)
The competition ELISA was carried out by coating with 50 µl of 10 µg/ml scFv fragment solution. After saturation, 100 µl of 5 µg/ml HP1ß-GST protein, which had been previously incubated for 30 minutes at room temperature with or without the purified anti-CD scFv fragments or the non-relevant anti-lysozyme D1.3 at a 1:10 molar ratio, was added to the coated wells. The HP1ß-GST bound protein was determined by further incubation with goat anti-GST antibodies (Amersham-Pharmacia) and rabbit anti-goat linked horseradish peroxidase (Pierce).
DNA constructs
For the expression in mammalian cells, all scFv fragments employed in this
work were subcloned into the NcoI/NotI sites of
pscFvexpress-cyt and pscFvexpress-nuc vectors
(Persic et al., 1997). For PCR
amplification of anti-CD scFv fragments from the bacterial pDAN-scFv vector,
the following degenerate primers were designed:
5'-GCAGCAAGCGGCGCCCATGGCC and 3'-TTTGGGATTGCGGCCGCGCTAGC. The
pscFvexp-cyt-163R4 (anti ß-gal), which was derived from pPM163-R4 (kindly
provided by P. Martineau), was subcloned as described previously
(Lener et al., 2000
).
Cell lines, transfection, immunoprecipitation and western
blotting
Simian COS fibroblasts, murine NIH-3T3 fibroblasts, human embryonal kidney
HEK-293 (kindly provided by R. Testi, University of Tor Vergata, Rome) and
human osteosarcoma Saos-2 p53 null cells (kindly provided by G. Melino,
University of Tor Vergata, Rome) were grown in DMEM medium supplemented with
10% fetal bovine serum.
COS and NIH-3T3 cells were transiently transfected as described previously
(Cardinale et al., 2001).
HEK-293 cells were transfected with Superfect (Qiagen) and Saos-2 cells with
lypofectamine 2000 reagent (Life Technologies) following the manufacturer's
instructions, with a DNA/transfectant reagent ratio (w/v) of 1:5 in both
cases. Z-VAD fmk (N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone)
(Vinci-Biochem) was used at a final concentration of 30 µM and added either
before or after transfection; it did not affect the rescue of apoptotic
cells.
Cells were harvested and analyzed 48 hours after transfection. Lysis,
extraction of cellular proteins from transfected cells, immunoprecipitation
and western blotting were performed as previously reported
(Cardinale et al., 2001). An
immunoblot of scFv fragments was performed using the anti-myc IgG 9E10
antibodies and immunoprecipitation by using the same affinity-purified
antibody linked to Protein-A Sepharose. Immunoblotting to detect HP1ß was
carried out with rat anti-HP1ß antibody MAC 353
(Wreggett et al., 1994
). The
following secondary antibodies were used: sheep anti-mouse IgG horseradish
peroxidase (Amersham-Pharmacia) and goat anti-rat IgG horseradish peroxidase
(Pierce).
Immunofluorescence
Immunofluorescence was carried out as described previously
(Lener et al., 2000).
Incubations with affinity-purified mouse anti-myc IgG 9E10, rat anti-HP1ß
IgG (MAC 353), rat anti-HP1
IgM (MAC 385), rat anti-M33 IgM (MAC 402)
and rabbit anti-HP1
IgG (M235) were performed at room temperature for 1
hour. Fluorescein-isothiocyanate-conjugated (FITC), goat anti-mouse IgG
(Pierce), Texas Red anti-mouse IgG (Calbiochem), FITC-conjugated goat anti-rat
IgG (Sigma), tetramethyl-rhodamine isothiocyanate (TRITC) rabbit anti-rat IgG
(Sigma), biotin-conjugated goat anti-rat IgM (Pierce), rhodamine-conjugated
goat anti-rabbit IgG (Sigma) and Texas-Red streptavidin (Amersham-Pharmacia)
were used for detection. The dye Hoechst 33258 was used at a 1 µg/ml
concentration. Samples were examined with a Leica fluorescence microscope and
CCD camera, equipped with a 100x oil immersion lens.
In situ identification of apoptotic cells
Apoptotic cells were visualized by staining with the blue fluorescent dye
Hoechst 33342 (Sigma) and a phosphatidylserine assay. Hoechst 33342 was used
at different concentrations, depending on the cell line: 0.3 µg/ml for COS,
0.25 µg/ml for NIH-3T3, 0.05 µg/ml for HEK-293 and 0.125 µg/ml for
Saos2 cells. Phosphatidylserine assay was performed using Annexin-V-FLUOS
staining kit (Boeringher-Roche) following the manufacturer's instructions.
Annexin-V- and Hoechst-33342-positive cells were counted from non transfected cells and cells transfected with different scFv fragments. The results shown in Figs 6 and 7 are the average from three different experiments for each cell line used. At least 50 positively transfected cells for each plasmid were counted and checked for their positive reaction to phosphatidylserine (Annexin V) and Hoechst 33342.
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Results |
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Recombinant anti-HP1ß scFv fragments were expressed in bacteria,
purified by nickel chelation chromatography and analyzed by western blotting
using the anti-tag SV5 monoclonal antibody (see Materials and Methods). As
shown in Fig. 2A, a major
immunoreactive band of expected size (32 kDa) is present (372scFv, lane 1;
373scFv, lane 2). A less abundant faster migrating band is also present in
each of the lanes, which probably represents a degradation product.
Coomassie-blue staining of the same gel shows no other contaminating bands,
indicating that the preparations are more than 95% pure. The two purified scFv
fragments were characterized for binding specificity using ELISA and shown to
maintain the same immunogenic properties of the original monoclonal antibodies
MAC 372 and MAC 373 (Fig. 2B).
Also, as shown in Fig. 2C, the
two scFv fragments recognize other CD proteins, namely HP1, mPc1 (M33)
and, to a lesser extent, HP1
. Binding is likely to be via the
chromodomain because scFv antibodies bind to a CD-GST fusion, albeit at a
reduced level when compared with the full-length HP1ß protein. The
reduced binding to the CD-GST fusion is most probably because this fusion
aggregates when stored in a purified form. This results in less soluble
material for coating of each ELISA well.
The scFv fragments bind non-overlapping epitopes within the CD
Using a competitive ELISA assay we next tested whether the scFv fragments
recognize different epitopes within the CD
(Fig. 2D). In this experiment,
ELISA plates were coated with each of the purified scFv fragments: 372 in
Fig. 2D, upper panel and 373
scFv fragment in Fig. 2D, lower
panel. The wells were next incubated with purified HP1ß-GST protein,
either on its own or after prior incubation with the purified anti-CD scFv
fragments or a non-relevant anti-lysozyme D1.3 scFv. The HP1ß-GST bound
by the scFv coating the wells was detected by further incubation with anti-GST
antibodies. As shown (Fig. 2D),
when HP1ß is preincubated with the same anti-CD scFv fragment used for
coating, the amount of HP1ß bound is significantly reduced. In contrast,
when HP1ß is preincubated with a different anti-chromodomain scFv
fragment from that coating the wells, or with the anti-lysozyme scFv, there is
no inhibition of the binding.
These data demonstrate that the scFv fragments bind non-competitively and therefore each recognizes a unique epitope within the HP1ß CD.
Expression of anti-chromodomain scFv fragments in mammalian
cells
The 372 and 373 scFv fragments were subcloned into vectors optimized for
intracellular expression in mammalian cells
(Persic et al., 1997). For
nuclear targeting, three nuclear localization signals (NLS) PKKKRKV of the
large T antigen of SV40 virus were tagged to the C-terminal of the scFvs, as
this signal has been shown to efficiently direct antibody fragments to the
nucleus (Biocca et al.,
1995
).
Immunofluorescence analysis of transiently transfected COS and NIH-3T3 fibroblasts revealed that the intracellular expression of anti-CD scFv fragments cause a cell-lethal phenotype. Most of the transfected cells are round and tend to detach from the dish, indicating that CD function is required for cell survival. This is not a general cytotoxic effect of intracellular antibodies because expression of scFv fragments against non relevant antigen, such as, for example, anti-ßgal, did not result in cell sickness (Figs 6 and 7). Notwithstanding this highly toxic effect, some transfected cells retain normal nuclear morphology, and this has allowed us to study the intracellular localization of the expressed scFv fragments. In particular, 372 anti-CD scFv fragments localize to the nucleus regardless of whether the scFv contained a NLS or not (Fig. 3A,C), giving rise to two major types of pattern a punctate pattern on the background of a more uniform staining (Fig. 3A) and another where there is only uniform staining of the nucleus with few nuclear aggregates (Fig. 3C). The percentage of cells displaying nuclear aggregates varies between 50 and 70 depending the cell line transfected. Nuclear localization was also obtained with the nuclear version of 373 (Fig. 3E), although 373cyt showed only a partial localization to the nucleus, with the bulk of 373cyt remaining in the cytoplasm (Fig. 3G).
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The simplest explanation for the observation that cytoplasmic 372 anti-chromodomain scFv can localize to the nucleus (Fig. 3C) is that the interaction between the intracellularly expressed scFvs and the NLS-containing HP1 proteins occurs in the cytosol. The antigen-antibody complex is then transported across the nuclear membrane into the nucleus because of the NLS present in HP1 proteins.
Coimmunoprecipitation of HP1 proteins and anti-chromodomain
scFvs
Guided by our observations we next wanted to investigate whether the scFvs
do, in fact, interact with HP1 proteins in vivo. In order to do this we
performed coimmunoprecipitation experiments. COS cells were transfected with
372-nuc and 373-nuc scFv fragments and analysed after 48 hours. The scFvs were
then immunoprecipitated from cell extracts using the anti-myc 9E10 monoclonal
antibody (see Material and Methods). After separation of the
immunoprecipitated proteins by SDS/PAGE, they were western blotted and probed
with the anti-HP1, ß,
and mPc1 monoclonal antibodies. In
an initial control experiment, where cells were transfected with an
anti-ßgal scFv, we found that, under these conditions, HP1ß was
mostly found in the insoluble fraction, with no detectable HP1ß in the
soluble fraction (Fig. 4A,
lanes 1 and 2). However, in extracts taken from cells expressing the
anti-chromodomain scFvs (372nuc, lane 3; 373nuc, lane 4), we found that
HP1ß could be detected in the soluble fraction. Thus, expression of the
intracellular scFvs results in the release of HP1ß from the insoluble
fraction, which is most probably caused by direct in vivo binding of the
intracellular scFvs to HP1ß. Moreover, despite the fact that scFvs
interact with all three HP1 proteins in vitro
(Fig. 1) we found that the
anti-chromodomain scFv fragments did not immunoprecipitate HP1
,
and mPc1 (data not shown), suggesting that the more accessible target of the
intracellular antibodies in living cells is the HP1ß CD.
|
We also undertook additional immunoblots using the anti-myc IgG to
determine the level of expression of anti-CD scFvs relative to scFvs directed
against a non-relevant antigen (ßgal). As shown in
Fig. 4B, the intracellular
anti-ßgal scFv fragment (lane 1) is expressed at a higher level with
respect to the anti-CD scFv fragments (lanes 2 and 3). The difference in
molecular weight between the anti-ßgal and the anti-CD scFv fragments
(372nuc and 373nuc) is because of the presence of three NLS at the C-terminal
of the anti-CD molecules. We also noted that, in addition to the weak anti-CD
scFv bands, other immunoreactive bands at lower and higher molecular mass are
also present (Fig. 4B, lanes 2
and 3). The lower bands represent degradation products, whereas the higher
bands are probably the result of post-translational modification of the
antibody fragments, such as ubiquitination
(Cardinale et al., 2001). The
marked reduction in the expression level is likely to reflect the general
state of the transfected cells, which, as already mentioned, appear sick and
some of them start to detach from the plate.
Diverting HP1ß into nuclear aggregates using intracellular
antibodies
We next explored the effect of intracellular scFvs expression on HP1
nuclear localization in living cells. We analyzed the effect of scFv 372nuc
because of the higher transfection efficiency achieved with this plasmid,
which allowed us to look at more cells. Accordingly, scFv 372nuc was
transfected into mouse NIH-3T3 fibroblasts, which were chosen because they
possess large Hoechst-positive heterochromatic blocks that are easily
observable with a fluorescence microscope: these blocks provide a `landmark'
against which HP1 localization could be measured. The steady-state
distributions of HP1 isoforms in NIH-3T3 cells was as expected
(Wreggett et al., 1994;
Horsley et al., 1996
;
Minc et al., 1999
). HP1
and HP1ß were found in large nucleoplasmic foci that largely colocalized
with Hoechst-positive heterochromatic blocks
(Fig. 5B,F for HP1ß;
Fig. 5J,N for HP1
).
HP1
labeling was different as it was distributed in multiple small dots
that were mainly located outside of the nucleoli and of the Hoechst-positive
heterochromatic blocks (Fig.
5R).
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Strikingly, we found that expression of the anti-CD 372nuc scFv fragment in some NIH-3T3 cells resulted in the formation of distinct HP1ß nuclear aggregates of different size, which were not coincident with Hoechst-positive heterochromatic blocks (Fig. 5A-D,E-H). As seen, the anti-myc antibody localizes to distinct foci (Fig. 5C,G), which are coincident with HP1ß foci (Fig. 5B,F). These coincident foci do not colocalize with the Hoechst-positive heterochromatic blocks (compare Fig. 5C,G with 5A,E). We conclude that these novel nuclear aggregates consist of HP1ß bound to the intracellular scFv.
We extended this study to investigate the effect of 372nuc scFv expression
on the localization of HP1, HP1
and mPc1. In cells where
expression of the intracellular antibody resulted in the formation of nuclear
aggregates, the localization of HP1
was not disturbed
(Fig. 5, see the nucleus in the
upper middle of panels K,L). Other cells, which showed a diffuse pattern of
scFv expression (Fig. 5, upper
nucleus in panel O), also exhibited a diffuse HP1
signal throughout the
nucleus (Fig. 5, upper nucleus
in panel N). Finally, in some cells the scFv fragment localized to the typical
HP1
heterochromatic foci (see lower nucleus in
Fig. 5, panels M-P). The
pattern of HP1
and mPc1 distribution did not change in cells expressing
the intracellular scFv (Fig. 5, panels Q-T for HP1
and panels U-X for mPc1).
These findings are largely consistent with the immunoprecipitation
experiments (Fig. 4) and
support the conclusion that the anti-CD scFv fragments interact almost
exclusively with the HP1ß isoform in vivo. This interaction then diverts
HP1ß, in an antibody-specific way, from its normal physiological location
(Fig. 5A-H). Whether the
diffuse distribution of HP1 seen in some nuclei is caused by a direct
in vivo interaction with the anti-CD scFvs or is the result of a general
perturbation of chromoproteins distribution, as cells are dying (see next
section), is being studied.
Chromodomain inactivation causes cell death
We noticed that cells expressing intracellular antichromodomain scFvs did
not thrive, with many of the cells exhibiting cell shrinkage, which is a
feature of apoptosis. In order to explore the cytotoxic effect of the
intracellularly expressed anti-chromodomain antibodies, we decided to quantify
the number of cells that undergo cell death by using two markers of apoptosis.
First, we used Annexin V, which detects alterations at the level of the plasma
membrane. Second, we used the blue-fluorescent Hoechst 33342 dye, which stains
the condensed chromatin of apoptotic cells more brightly than the chromatin of
non-apoptotic cells. On the basis of the combined staining patterns of these
dyes, we were able to distinguish between normal, apoptotic and dead
cells.
We transiently transfected mouse NIH-3T3, simian COS fibroblasts and human HEK-293 cells and determined the phenotype of transfected cells using the two markers of apoptosis. As seen in Fig. 6A,B the percentage of apoptotic cells varies between 80-90% in COS and HEK-293 and 95-98% in mouse NIH-3T3 fibroblasts, when calculated on the basis of Annexin-V-positive cells, and between 70-90% on the basis of Hoechst-33342-positive cells. Only very few Annexin-V/Hoechst-positive cells are also positive for propidium iodide (not shown), indicating that the cells examined are not necrotic but are actually undergoing a process of programmed cell death. The cytoplasmic versions of the anti-CD scFvs are equally as efficient in inducing apoptosis as the nuclear scFvs (Fig. 6). This observation suggests that the cytoplasmic scFvs interact with HP1 proteins in the cytoplasm and that this interaction may interfere with their normal intracellular traffic. By contrast, much lower levels of apoptosis were observed in cells transfected with the anti-ßgal scFv constructs.
Cell death induced by anti-CD scFv is p53 independent and is only
partly prevented by Z-VAD fmk treatment
We next investigated whether the cell death seen in our experiments was
dependent on p53 tumor-suppressor gene activity because p53 is activated in
response to a variety of signals of DNA damage
(Levine, 1997). Human
osteosarcoma Saos-2 cells (Chen et al.,
1990
), which are null mutants for p53, were transiently
transfected with the different anti-CD scFv fragments.
Fig. 7A shows that over 90% of
Saos-2 cells transfected with 372nuc or 373nuc plasmid undergo cell death,
suggesting that cell death occurs in a p53 independent pathway. In the control
experiments, where Saos-2 cells were transfected with an irrelevant
anti-ßgal scFv fragment or were not transfected, only 10% of the cells
undergo apoptosis.
The role of caspases (cysteine proteases cleaving after particular
aspartate residues) in anti-CD scFv-dependent apoptosis was investigated by
treating cells with Z-VAD fmk, a wide spectrum inhibitor of caspases
(Ekert et al., 1999). We
therefore treated NIH-3T3 cells with Z-VAD fmk after transfecting them with
372nuc and 373nuc scFv fragments. As seen in
Fig. 7B there was a small, but
significant effect of Z-VAD fmk on cell death that results from the expression
of anti-CD scFvs (around 20-25% decrease of cell death). By contrast, the
caspase inhibitor is able to rescue more than 50% of non transfected cells
treated with 250 µM H2O2. The cytoplasmic version of
the two anti-CD scFv fragments was equally effective in inducing cell death
when transfected into Saos-2 p53-/-, and this effect was only
partly rescued by Z-VAD fmk treatment in NIH-3T3 cells (data not shown).
We conclude that the anti-chromodomain intracellular antibody fragments induce cell death via a pathway that is p53 independent and only partly caspase dependent.
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Discussion |
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Despite the broad in vitro specificity of the scFvs, immunoprecipitation
experiments revealed that the scFvs in vivo target was more limited and
probably restricted to HP1ß (Fig.
4). This was supported by subnuclear localization studies, which
showed that, just before transfected cells begin to exhibit signs of
apoptosis/cell death, HP1ß was diverted from its normal nuclear
localization into intranuclear aggregates
(Fig. 5A-H). Diversion from
typical steady-state nuclear localization was not observed for HP1, M32
(HP1
) or mPc1, which contain highly homologous, but non-identical, CDs.
Nevertheless, we cannot rule out the possibility that other as yet
unidentified CD proteins might be recognized by the anti-CD scFvs, and the
inactivation of CD could contribute to the observed cell death phenotype.
The nuclear aggregates observed (Fig.
5A-H) closely resemble previously described cytoplasmic aggresomes
that result from the intracellular expression of scFvs. These cytoplasmic
aggresomes contain ubiquitinated scFv fragments as a complex with trapped
antigen (Cardinale et al.,
2001): the anti-chromodomain scFvs also appear to contain
modified, possibly ubiquitinated, antigen-antibody complexes
(Fig. 4B). The formation of
nuclear aggregates are consistent with our previous work, which has suggested
that intracellular scFvs act by interfering with intracellular traffic of
target proteins, leading to mis-localization away from their normal
physiological sites of action (Lener et
al., 2000
). The efficacy of the cytoplasmic scFvs can also be
explained by a similar mechanism where newly synthesized HP1 molecules are
inhibited or slowed down in their translocation towards the nucleus as they
become bound by the scFvs (Fig.
5). Studies presently underway to characterize the intranuclear
aggregates described in this paper will shed light on their possible
similarities with cytoplasmic aggresomes, that is, to verify whether they are
ubiquitinated and whether they are associated with nuclear proteasomes.
Clearly defined nuclear aggregates are not visible in all cells. In some cells the anti-chromodomain scFvs and HP1 proteins are diffuse, and a widespread fluorescence signal is distributed throughout the nucleoplasm (Fig. 3C; Fig. 5O). We suspect that this uniform distribution is an indirect effect of cell death, because it appears mostly in cells that present early signs of apoptosis, as determined by Annexin V staining. Moreover, immunolocalization of HP1 proteins in cells undergoing apoptosis as a result of exposure to H2O2 show a significant delocalization of these proteins in the nucleoplasm (I.F. and S.B., unpublished).
The in vivo preference of anti-chromodomain scFvs for HP1ß may reflect
the fact that the epitope in the HP1ß chromodomain is simply more
accessible to the scFvs in bulk chromatin. In addition, the preference may
reflect the observation that HP1ß shows dynamic changes in nuclear
localization during the cell cycle (Minc
et al., 1999; Kourmouli et
al., 2000
), and it is this dynamic behavior that allows the
intracellular scFvs to bind and sequester the HP1ß in the nuclear
aggregates. Recent work using isotype-specific antibodies showed that, despite
their being similar in their primary sequence, HP1 isotypes segregate into
distinct nuclear domains (Minc et al.,
1999
). In particular, during mitosis, HP1ß detaches from
heterochromatin, becomes cytoplasmic and only later re-assembles onto the
polar surfaces of anaphase chromosomes
(Kourmouli et al., 2000
). To a
lesser degree, HP1
also shows dynamic behavior during the cell cycle;
HP1
largely remains on the chromosomes
(Minc et al., 1999
;
Kourmouli et al., 2000
).
What physiological function of HP1ß is perturbed by the intracellular
scFvs and thus leads to the dramatic cell death phenotype? One possibility is
that the intracellular antibodies hinder the assembly and/or maintenance of
heterochromatin complexes. The perturbation of such complexes could lead to a
variety of lesions, each of which is likely to be incompatible with cell
survival. They include activation of normally silent genes
(Jones et al., 2000), defects
in centromere function (Kellum and
Alberts, 1995
), ectopic telomere fusions (Fanti et al., 1999) and
inappropriate NE re-assembly (Kourmouli et
al., 2000
). In particular, we note that HP1 proteins interact with
the non homologous end joining (NHEJ) protein Ku70
(Song et al., 2001
), which has
been found to play an important role as part of a complex with Sir3 and Sir4
in maintaining genome integrity in budding yeast
(Guarente, 1999
). By analogy,
mammalian HP1 proteins may serve a similar role to Sir3 and Sir4 in
maintaining structured chromosomal domains that are necessary for cell
survival. Interference with this function could lead to catastrophic effects
on genome integrity. Another site where perturbation of HP1 function is likely
to have a dramatic effect is at the centromere. The centromere is known to
harbor proteins that are necessary for cell survival. The best known being
survivin (Li et al.,
1998
; Li et al.,
1999
). Loss of survivin activity leads to irreversible cell death,
and we suspect that anti-chromodomain scFvs may activate this pathway.
Whatever the mechanism, our results provide evidence that scFvs directed
against distinct epitopes of the HP1 chromodomain induce cell death when
expressed in mammalian cells, and this result strengthens the hypothesis that
there is an absolute requirement for HP1 chromodomain function in mammalian
cells.
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Acknowledgments |
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References |
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