Download Progeria, the nucleolus and farnesyltransferase inhibitors

Nuclear Envelope Disease and Chromatin Organization 2009
Progeria, the nucleolus and farnesyltransferase
inhibitors
Ishita S. Mehta*, Joanna M. Bridger* and Ian R. Kill†1
*Laboratory of Nuclear and Genomic Health, Centre for Cell and Chromosome Biology, Division of Biosciences, School of Health Sciences and Social Care,
Brunel University, Uxbridge UB8 3PH, U.K., and †Laboratory of Cellular Gerontology, Centre for Cell and Chromosome Biology, Division of Biosciences,
School of Health Sciences and Social Care, Brunel University, Uxbridge UB8 3PH, U.K.
Abstract
HGPS (Hutchinson–Gilford progeria syndrome) is a rare genetic disease affecting children causing them to
age and die prematurely. The disease is typically due to a point mutation in the coding sequence for the
nuclear intermediate-type filament protein lamin A and gives rise to a dominant-negative splice variant
named progerin. Accumulation of progerin within nuclei causes disruption to nuclear structure, causes and
premature replicative senescence and increases apoptosis. Now it appears that accumulation of progerin
may have more widespread effects than previously thought since the demonstration that the presence
and distribution of some nucleolar proteins are also adversely affected in progeria cells. One of the major
breakthroughs both in the lamin field and for this syndrome is that many of the cellular defects observed
in HGPS patient cells and model systems can be restored after treatment with a class of compounds known
as FTIs (farnesyltransferase inhibitors). Indeed, it is demonstrated that FTI-277 is able to completely restore
nucleolar antigen localization in treated progeria cells. This is encouraging news for the HGPS patients who
are currently undergoing clinical trials with FTI treatment.
HGPS (Hutchinson–Gilford progeria
syndrome)
HGPS is an extremely rare disorder that affects children,
causing them to age prematurely. Clinical features of this disease include alopecia, growth retardation, an aged appearance,
loss of subcutaneous fat, progressive atherosclerosis, bone
deformities and cardiovascular diseases [1]. HGPS is typically
caused by an autosomal dominant de novo mutation in the
LMNA gene that encodes the nuclear intermediate filament
proteins lamins A and C [2,3], both of which are components
of the nuclear lamina (a structure that has roles in DNA replication, transcription, chromatin organization and remodelling,
maintenance of nuclear shape and integrity and cell division)
[4]. The most common mutation associated with HGPS is
a single base-substitution in codon 608 of exon 11 on the
LMNA gene, which results in the formation of a cryptic splice
site producing a truncated prelamin A protein (progerin) that
lacks 50 amino acids within the C-terminus end. Progerin
acts in a dominant-negative manner regardless of whether its
expression occurs naturally through alternative splicing or it
is activated using anti-sense oligonucleotides [5]. This single
base-pair mutation not only leads to disruption of the nuclear
lamina and nuclear shape but also has perhaps more clinically
relevant effects on major processes taking place in the cell
nucleus, such as the epigenetic control of chromatin, gene
Key words: farnesyltransferase I inhibitor, fibrillarin, Hutchinson–Gilford progeria syndrome
(HGPS), Ki67, nucleolus.
Abbreviations used: DFC, dense fibrillar component; FC, fibrillar centre; FTI, farnesyltransferase
inhibitor; HGPS, Hutchinson–Gilford progeria syndrome; rDNA, ribosomal DNA.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2010) 38, 287–291; doi:10.1042/BST0380287
expression, cell division, interphase chromosome positioning,
DNA replication and the DNA damage response [4,6,7].
In normal cells, prelamin A, a precursor protein from
which mature lamin A is derived, contains a CaaX (where
a is an aliphatic residue) motif at the C-terminal end that
signals farnesylation of the cysteine residue by the enzyme
farnesyltransferase [8]. The presence of a farnesyl group at
the C-terminal end along with the CaaX motif promotes the
association of prelamin A with the nuclear membrane and
is needed for correct localization of the mature protein [9].
The protein then loses the three terminal aaX amino acids
followed by methyl esterification of the C-terminal cysteine
residue. Finally, the protein undergoes endoproteolytic cleavage by an enzyme ZMPSTE24 (FACE1) metalloproteinase
[10], resulting in a loss of 15 amino acids at the C-terminal
end, including the farnesylated cysteine, thus generating
mature lamin A protein [11]. The final step of this posttranscriptional modification process whereby the farnesyl
group is cleaved is thought to be important for insertion of
the mature lamin A into the nuclear lamina [12]. In progerin, the deletion of 50 amino acids within the C-terminus
does not affect the CaaX motif and the protein undergoes
normal farnesylation. However, the endoproteolytic site
normally cleaved by ZMPSTE24 is missing and hence
progerin remains permanently farnesylated [13]. Retention
of the farnesyl group and accumulation of the farnesylated
protein at the nuclear envelope are thought to compromise
nuclear integrity, disrupt the nuclear lamina including the
underlying heterochromatin and lead to the formation of
abnormally shaped nuclei, a prominent characteristic seen in
HGPS [14,15]. Thus a concept that blocking farnesylation of
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Figure 1 Presence and distribution of nucleolar proteins in control and HGPS fibroblasts
Control fibroblasts (2DD) and fibroblast cell strains derived from three HGPS patients, namely AG01972, AG11513 and
AG11498, were cultured on sterile glass coverslips in 15 % (v/v) foetal bovine serum+Dulbecco’s modified Eagle’s medium
until the cultures became senescent. Cell samples were collected from early and late cell passages and fixed with 4 %
(w/v) paraformaldehyde followed by permeabilization with methanol/acetone (1:1). Mid-passage HGPS cultures were
subjected to a 48 h incubation in 2.5 μM FTI-277 (Sigma). Dual-colour indirect immunofluorescence was employed to assess
the distribution of pKi67 [rabbit anti-Ki67 antibody (Novacastra); 1:1500 dilution], nucleolin [mouse anti-nucleolin antibody
(Abcam); 1:200 dilution] and fibrillarin (mouse anti-fibrillarin antibody; 1:1000 dilution). Anti-pKi67 staining was revealed
by using swine anti-rabbit antibody (Jackson Immunoresearch Laboratories) conjugated to FITC (green) (A, C, E, G, I, K, M,
O, Q, S, U, W); other anti-nucleolar staining was revealed by using goat anti-mouse antibody (Dako) conjugated to TRITC
(tetramethylrhodamine β-isothiocyanate) (red) (fibrillarin, B, D, F, H, J, L; nucleolin, N, P, R, T, V, X). Greyscale images were
acquired on an Olympus BX microscope using Smartcapture 3 software and a Model viewpoint GS greyscale digital camera.
Scale bar, 10 μM.
progerin might help ameliorate disease pathology seen in
HGPS cells was put forward in 2003, shortly after the discovery of the gene involved in causing HGPS. To test this hypothesis, a class of drugs called FTIs (farnesyltransferase inhibitors) were used. FTIs inhibit attachment of a farnesyl group
to proteins by irreversibly binding to the CaaX domain [16].
FTI treatment for HGPS patients
Encouragingly, the first few studies demonstrated that
treating HGPS patient cells in culture with FTI prevented an
accumulation of progerin at the nuclear envelope and reduced
the frequency of abnormally shaped nuclei [12,17–20]. This
finding was also demonstrated in HeLa HGPS model cells
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Authors Journal compilation [18] and in culture in fibroblasts derived from a mouse model
[20]. A study by Glynn and Glover [17] demonstrated an
improved morphology and a reduction in percentage of
abnormal nuclei, by 70 % in HGPS cells after treatment with
low doses of FTIs (10 nM), over a 14 day period. Significant
dose-dependent reduction in nuclear blebbing, as well as
redistribution of mutant protein, was reported within 48–72 h
of FTI treatment on HGPS cells [12], ZMPSTE24−/− mouse
embryonic fibroblasts [19] and LmnaHG/+ and LmnaHG/HG
mouse fibroblasts [20]. HGPS cells treated with FTIs for
72 h also show improved nuclear stiffness to levels almost
comparable with normal cells and significant restoration of
directional persistence with regard to cell migration and thus
improvements in wound healing ability [21]. However, FTI
Nuclear Envelope Disease and Chromatin Organization 2009
Table 1 The fraction of cells displaying typical and atypical distributions of fibrillarin in proliferating and non-proliferating normal and
HGPS cells, before and after FTI treatment
Staining
Ki67-positive
Ki67-negative
Cells
Typical
nucleolar
Atypical
nucleolar
Negative
Typical
nucleolar
2DD early passage
2DD late passage
43
7
0
0
0
0
57
93
AG01513 early passage
AG01513 late passage
AG01972C early passage
59
11
65
0
0
0
0
0
0
AG01972C late passage
AG01148 early passage
AG01148 late passage
5
66
5
0
0
0
AG11498+FTI
29
0
Negative
Total typical
nucleolar (%)
0
0
0
0
100
100
0
0
0
41
90
33
0
0
0
59
11
65
0
0
0
0
0
0
95
34
95
0
0
0
5
66
5
0
23
48
0
52
treatment was unable to restore the DNA damage response
mechanism, which is affected in HGPS cells [22].
With promising results from in vitro studies with regard to
the ability of FTIs to reverse nuclear abnormalities, various
laboratories then focused on animal models of HGPS to test
these drugs further. The most common models available for
progeria are ZMPSTE24−/− and LmnaHG/+ mouse models.
Treatment of ZMPSTE24−/− mice with FTI, beginning at
5 weeks of age, showed the presence of non-farnesylated
prelamin A, improved growth and survival rates, better bone
integrity and increased grip strength [23]. In LmnaHG/+ mice,
also treated with FTIs, there is an increase in survival rates,
an increased body weight (adipose) and reduced rib fractures
[24,25]. A more recent study that uses a transgenic mouse
model carrying the human G608G LMNA mutation and
displaying a cardiovascular phenotype, a characteristic reason
that causes death in most HGPS patients, demonstrated that
FTI treatment reduces VSMC (vascular smooth-muscle cell)
loss and proteoglycan accumulation and thus retards the
onset, as well as progression, of cardiovascular diseases in
these mice [26].
The studies have so far been so successful that a collection
of progeria children have been placed on a regime of FTIs
[27,28] the results of which will be revealed in 2010.
Nucleoli in HGPS cells before and after FTI
treatment
The studies have so far been concentrated on the defects of the
nuclear lamina and functions related to the lamina in HGPS
cells. Since lamin A is not only present at the nuclear lamina
but is also a component of the nuclear matrix [29–31] and is
found within the nuclear interior [32], with sites of replication
[33], transcription factories [34] and splicing speckles [35],
mutations in the LMNA gene might affect other nuclear structures and bodies. In particular, we wondered whether impaired lamin A function may affect the structure and/or func-
Atypical
nucleolar
tions of the nucleolus. It has been demonstrated recently that
lamin B1 does indeed play a role in nucleolar integrity [36].
The nucleolus is a membraneless, crucial nuclear compartment involved in ribosomal biogenesis. Ultrastructural
analysis of nucleoli has defined three subcompartments: the
FCs (fibrillar centres), the DFC (dense fibrillar component)
and the granular component [37], each of which has distinct
but related functions [38]. Some nucleolar proteins are
constrained within these nucleolar compartments; for
example, fibrillarin and Ki67 are located in the DFC [39,40],
nucleophosmin-B23 is localized in the granular component
[41,42], and RNA polymerase I is localized within the
FC [43,44]. Apart from transcription of rDNA (ribosomal
DNA) genes and processing of pre-ribosomal particles,
the nucleolus is also involved in many diverse fundamental
cellular processes [45].
In order to question whether nucleolar structure was
affected in HGPS cells, both normal and HGPS fibroblasts
were stained with antibodies reacting with pKi67, nucleolin
and fibrillarin. Each of these antigens has a distinct role
in nucleolar function but all three occupy regions of the
DFC. Ki67 is present only in proliferating cells and is
therefore a robust marker of both proliferative state and
nucleolar integrity [40]. Nucleolin is involved in a number of
processes including rDNA transcription, rRNA processing,
nucleocytoplasmic transport and regulation of apoptosis [46].
Fibrillarin is involved in pre-rRNA processing and ribosome
assembly [47,48]. Representative images of staining patterns
in normal and HGPS cells are shown in Figure 1. Ki67 staining
in both control and HGPS cells appeared normal with typical
staining patterns revealed in all Ki67-positive (proliferating)
nuclei (Figures 1A, 1E, 1I, 1M, 1Q and 1U). In these Ki67positive cells, staining for both fibrillarin (Figures 1B, 1F
and 1J) and nucleolin (Figures 1N, 1R and 1V) appeared
normal. However, in Ki67-negative cells (non-proliferating),
staining patterns for fibrillarin in control cells were normal
(Figure 1D), and in HGPS cells, they appeared abnormal
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Table 2 The fraction of cells displaying typical and atypical distributions of nucleolin in proliferating and non-proliferating normal and
HGPS cells, before and after FTI treatment
Staining
Ki67-positive
Cells
Typical
nucleolar
2DD early passage
2DD late passage
44
4
AG01513 early passage
AG01513 late passage
AG01972C early passage
Ki67-negative
Atypical
nucleolar
Negative
Typical
nucleolar
Atypical
nucleolar
0
0
0
0
46
95
10
1
0
0
90
99
68
17
44
6
2
7
1
0
1
12
6
0
9
58
48
0
9
11
80
23
44
AG01972C late passage
AG01148 early passage
AG01148 late passage
1
34
1
1
26
2
0
0
0
0
2
0
93
34
79
41
4
26
1
46
1
AG11498+FTI
30
0
0
59
10
0
89
with reduced intensity and lack of any punctate structure
(Figure 1H). Similarly, staining for nucleolin in Ki67-negative
cells was normal in control cells (Figure 1P) but abnormal in
HGPS cells (Figure 1T). Tables 1 and 2 show the percentages
of cells displaying typical, atypical and negative staining for
fibrillarin and nucleolin in Ki67-positive and Ki67-negative
cells for normal fibroblasts (2DD) and three HGPS fibroblast
cultures. The table includes data for both early passage and
late passage cultures, showing how the relative proportions
of staining class change with progression of replicative
senescence. We have previously shown that with an increase in
cellular age, cellular abnormalities predominate within HGPS
cultures [49]. Indeed, atypical staining for both nucleolin and
fibrillarin in Ki67-negative cells increases between early and
late passage for all three HGPS cultures. Furthermore, the
proportion of cells with an absence of nucleolin staining
increases with cellular age in HGPS cultures, whereas the
absence of nucleolin staining is never observed in control
cultures. The total proportion of cells (Ki67-positive plus
Ki67-negative) displaying typical nucleolar staining for both
fibrillarin and nucleolin remains high in early and late passage
control cells. In contrast, there is a dramatic decline in the proportion of typically stained cells in all three HGPS cultures
with an increase in cellular age for both fibrillarin and nucleolin. Remarkably, the proportion of HGPS cells displaying
typical nucleolar staining for both fibrillarin and nucleolin is
partially or fully restored respectively after treatment for 48 h
with 2.5 μM of the FTI, FTI-277 (Figures 1L and 1X).
It seems that accumulation of progerin in cells may have
even more widespread consequences than already discussed in
the literature. The observations reported here that certain aspects of nucleolar structure are also altered in HGPS cells may
provide further insight into the cellular pathology of the disease. On a positive note, it appears that FTI treatment can effectively restore these nucleolar abnormalities, a finding that
provides more encouragement for the efficacy of FTI treatments currently undergoing clinical trials on HGPS patients.
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Total typical
nucleolar (%)
Acknowledgement
We thank Dr John Aris (University of Florida, Gainesville, FL, U.S.A.)
for the monoclonal anti-fibrillarin antibody.
Funding
We thank the Brunel Progeria Research Fund for financial support
of the present studies. I.S.M. is partially funded by an Overseas
Research Student Award Scheme.
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Received 3 August 2009
doi:10.1042/BST0380287
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C 2010 Biochemical Society
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