Molecular Biology of the Cell
Vol. 19, 5238 –5248, December 2008
Suppression of Proliferative Defects Associated with
Processing-defective Lamin A Mutants by hTERT
or Inactivation of p53
Brian A. Kudlow,* Monique N. Stanfel, Christopher R. Burtner,
Elijah D. Johnston, and Brian K. Kennedy
Department of Biochemistry, University of Washington, Seattle, WA 98195
Submitted May 19, 2008; Revised September 12, 2008; Accepted October 1, 2008
Monitoring Editor: Wendy Bickmore
Hutchinson-Gilford progeria syndrome (HGPS) is a rare, debilitating disease with early mortality and rapid onset of
aging-associated pathologies. It is linked to mutations in LMNA, which encodes A-type nuclear lamins. The most frequent
HGPS-associated LMNA mutation results in a protein, termed progerin, with an internal 50 amino acid deletion and,
unlike normal A-type lamins, stable farnesylation. The cellular consequences of progerin expression underlying the
HGPS phenotype remain poorly understood. Here, we stably expressed lamin A mutants, including progerin, in otherwise
identical primary human fibroblasts to compare the effects of different mutants on nuclear morphology and cell
proliferation. We find that expression of progerin leads to inhibition of proliferation in a high percentage of cells and
slightly premature senescence in the population. Expression of a stably farnesylated mutant of lamin A phenocopied the
immediate proliferative defects but did not result in premature senescence. Either p53 inhibition or, more surprisingly,
expression of the catalytic subunit of telomerase (hTERT) suppressed the early proliferative defects associated with
progerin expression. These findings lead us to propose that progerin may interfere with telomere structure or metabolism
in a manner suppressible by increased telomerase levels and possibly link mechanisms leading to progeroid phenotypes
to those of cell immortalization.
INTRODUCTION
The nuclear lamins are broadly expressed and constitute the
only intermediate filament proteins localized to the nucleus
(Smith et al., 2005; Bridger et al., 2007; Dechat et al., 2008).
Given the ability of lamins to form stable polymers resulting
from coiled-coil interactions, they have typically been ascribed a structural role in the nucleus (McKeon et al., 1986;
Lammerding et al., 2004, 2006); however, a variety of regulatory roles have also been proposed (Spann et al., 1997;
Spann et al., 2002; Frock et al., 2006; Ivorra et al., 2006; Nitta
et al., 2006, 2007; Scaffidi and Misteli, 2008). The two classes
of lamins, B-type and A-type (of which lamin A and lamin C
are the most prominent), have different patterns of expression. Whereas, isoforms of lamin B are expressed in all cells
throughout development, lamins A and C (lamin A/C) are
not expressed early in development. Instead, their expression begins during midgestation and is most prominent in
differentiating and terminally differentiated cells (Rober
et al., 1989).
Mutations in LMNA (encoding all A-type lamins) are associated with at least 12 different human genetic disorders
(Kudlow et al., 2007). Of these, at least three have features
This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 – 05– 0492)
on October 8, 2008.
* Present address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347.
Address correspondence to: Brian K. Kennedy (bkenn@u.
washington.edu).
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reminiscent of premature aging. The most common LMNAassociated premature aging-like syndrome, Hutchinson-Gilford progeria syndrome (HGPS), is often caused by a single
nucleotide change in exon 11 of the LMNA gene (Cao and
Hegele, 2003; De Sandre-Giovannoli et al., 2003; Eriksson et
al., 2003). This mutation, generally referred to as G608G, is
silent with regard to the coded amino acid but drives aberrant splicing of the lamin A pre-mRNA and ultimately results in the production of a mutant lamin A protein, termed
progerin, that contains a 50-amino acid deletion within its C
terminus. Other less frequent mutations in LMNA are also
associated with progeroid syndromes (Eriksson et al., 2003;
Kudlow et al., 2007).
Lamin A is synthesized as a 664-amino acid precursor
protein called prelamin A. This protein contains a C-terminal CaaX motif that directs its farnesylation (Lutz et al., 1992;
Sinensky et al., 1994). The farnesylated prelamin A protein
then undergoes two cleavage events that successively remove amino acids, including the prenylated cysteine-residue, from the C terminus to produce mature lamin A. Lamin
C has a divergent C terminus that lacks the CaaX motif and
therefore avoids these processing events. Progerin retains
the C-terminal CaaX motif normally found in prelamin A;
however, the sequences required for complete processing to
mature lamin A are lost in its 50 amino acid deletion. As a
result, progerin, unlike mature lamin A, is stably farnesylated. Several lines of evidence suggest that the stable farnesylation of progerin contributes to its toxicity and may
partially underlie the disease phenotypes associated with
expression of this mutant protein (Capell et al., 2005;
Mallampalli et al., 2005; Varela et al., 2005; Fong et al., 2006;
Yang et al., 2006).
Supplemental Material can be found at:
© 2008 by
http://www.molbiolcell.org/content/suppl/2008/10/08/E08-05-0492.DC1.html
The American Society for Cell Biology
hTERT Suppresses Progerin-linked Defects
Considerable progress has been made in understanding
the phenotypes of HGPS cells by studying patient-derived
fibroblasts or cells from mouse models of HGPS. However,
in the case of patient-derived fibroblasts, no isogenic control
cell lines are available. Furthermore, these cells have been
moved from tissue biopsies to culture, and it is unclear
whether the phenotypes observed are direct results of progerin expression or secondary results either of cell non-autonomous changes in vivo or adaptation of the progeria cells
to in vitro culturing. Although several important findings
have been made using patient fibroblasts (Scaffidi and
Misteli, 2005; Verstraeten et al., 2008), we wanted to develop
isogenic primary fibroblasts that would permit observation
of both the immediate and long-term consequences of progerin expression. Here, we describe the derivation and characterization of these cells, and we compare them to cells
expressing other lamin A mutants. Our findings indicate
that expression of progerin or a stably farnesylated lamin A
processing mutant causes proliferation defects that are rescued either by stable expression of hTERT or by inactivation
of p53.
MATERIALS AND METHODS
Plasmids
Expression of wild-type lamin A, progerin, and L647R mutant lamin A
proteins was directed by the retroviral vector pMXIH, as described previously
(Kudlow et al., 2005). A cDNA encoding progerin with its CaaX motif mutated
to SaaX was generated in pBluescript by site-directed mutagenesis. This
mutant was termed progerin-CS and was subcloned into the BamHI and XhoI
of pMXIH. pLXSN-hTERT, pLXSN-E6, and pLXSN-E7 were kind gifts of
Denise Galloway (Fred Hutchinson Cancer Research Center). A separate
retroviral vector, pBlox-TSH (Rubio et al., 2002), for hTERT expression was a
kind gift of Judy Campisi (Lawrence Berkeley National Laboratory).
Retroviral Transduction
Cell lines used were 82-6 primary dermal fibroblasts, provided by Junko
Oshima (University of Washington), AG13353 normal dermal fibroblasts
(Coriell Cell Repositories), AG01972C Hutchinson-Gilford patient fibroblasts
(bearing the G608G mutation), and 293T cells. In all cases, cells were cultured
in DMEM supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 10
U/ml penicillin, and 10 g/ml streptomycin. For expression of lamin mutants, 300,000 cells were infected three times in 36 h with amphotropic
retrovirus produced as described previously (Kudlow et al., 2005). In all cases,
cells were selected with 100 g/ml hygromycin-B for 7 d. For double infections, cells were first infected with the indicated pLXSN-based vector, twice in
24 h. Cells were selected for 10 d in 500 g/ml G418 (Invitrogen, Carlsbad,
CA). After G418 selection, cells were passaged twice, and then they were
reinfected with the appropriate pMXIH-based vector as described.
Scoring Colony Size, Plating Efficiency, and Nuclear
Morphology
Colony size-distribution assays were performed by plating cells at a density
of ⬃100 cells per 10-cm plate. Cells were then allowed to grow undisturbed
for 6 d. Plates were visually scanned and the number of cells per clone was
counted. Representative data were collected for between 74 and 148 clones. A
one-tailed Wilcoxon rank sum test was used to calculate the p value for the
hypothesis that the clone-size distribution of a given sample is skewed
toward smaller clones when compared with the clone-size distribution of the
relevant vector control. To determine plating efficiency, cells were plated at a
density of ⬃100 cells per 10-cm plate. Cells were allowed to grow undisturbed. At 2 or 6 d, cells were fixed with alcohol-Formalin-acetic acid (AFA;
70% ethanol, 3.7% formaldehyde, and 5% glacial acetic acid) and stained with
1% methylene blue in phosphate-buffered saline (PBS). Plates were then
visually scanned and the total number of clones, and cells per clone were
counted. Representative data were collected for five plates for each culture
and time point. Nuclear morphology was scored by staining cells with 4,6diamidino-2-phenylindole (DAPI) for DNA and antibodies for lamin B1 or
lamin A/C (see below). Cells treated with the farnesyltransferase inhibitor
(FTI) R115777 (provided by Michael Gelb, University of Washington) were
cultured in the drug for 48 h. Cells with oval nuclei were scored as “normal,”
whereas nuclei with protrusions, invaginations, sharp edges or blebs were
considered to be “dysmorphic.”
Vol. 19, December 2008
RNA Isolation, Reverse Transcription, Quantitative
Polymerase Chain Reaction (PCR), and Data Analysis
Cells (between population doubling [PDL] 1 and PDL 3) from two 10-cm
plates were harvested 24 h after feeding at ⬃50% confluence. RNA from six
independent cultures for each sample was isolated on different days with the
Ambion RNAqueous kit according to the manufacturer’s instructions. RNA
was treated with DNAse (Ambion) and used for reverse transcription reactions by using oligo(dT) primers and the SuperScript II kit from Invitrogen.
Quantitative PCR (qPCR) was performed to measure the expression level of
P21 (forward, 5⬘-GGCAGACCAGCATGACAGAT; reverse, 5⬘-GGACTGCAGGCTTCCTGTG), GADD45B (forward, 5⬘-GGTGGAGGAGCTTTTGGTGG; reverse, 5⬘-CAGAGGACCACGCTGTCTG), and IGFBP3 (forward, 5⬘GCATGCAGAGCAAGTAGACG; reverse, 5⬘-TAGCCAGCTGCTGGTCATGT).
We used three housekeeping genes for normalization: ␥-ACT (forward, 5⬘-GCTTGTATCTGATATCAGCACTGG; reverse, 5⬘-GAAAGGAAACTGGGTCCTACG),
GAPDH (forward: 5⬘-AAGAAGGTGGTGAAGCAGGCG; reverse, 5⬘-ACCAGGAAATGAGCTTGACAA), and TBP (forward, 5⬘-TTCGGAGAGTTCTGGGATTGTA; reverse, 5⬘-TGGACTGTTCTTCACTCTTGGC). Mock reverse transcribed
samples were used to verify that DNase treatment was completely effective. All reactions were performed in triplicate on an iCycler machine
using SYBRGreen mix (both from Bio-Rad) according to the manufacturer’s specifications. To normalize for mRNA input in each reaction, the
relative expression values for P21, GADD45B, and IGFBP3, were normalized to the geometric means of the expression value of the three housekeeping genes. Expression values for each gene are expressed as the
mean ⫾ SD of six independent RNA isolations. p values were calculated
with a two-tailed paired t test.
Senescence-associated -Galactosidase Activity and
Terminal Deoxynucleotidyl Transferase dUTP Nick-End
Labeling (TUNEL) Staining
Subconfluent fibroblasts were fixed in 3% formaldehyde and stained overnight at 37°C with 5-bromo-4-chloro-3-indolyl--d-galactoside buffered with
sodium phosphate at pH 6.0 in the presence of potassium ferrocyanide and
potassium ferricyanide as described previously (Dimri et al., 1995). Cells
staining robustly blue were counted as positive for senescence-associated
-galactosidase activity. Apoptotic cells were quantified by TUNEL staining
at PDL 2 with the Apo-5-bromo-2⬘-deoxyuridine TUNEL Assay kit (Invitrogen) according to the manufacturer’s directions but with minor adaptations
for adherent cells on coverslips. TUNEL-stained nuclei were counterstained
with DAPI, and TUNEL-positive nuclei were identified on an Axiovert 200
microscope (Carl Zeiss, Thornwood, NY). As a positive control, cells were
treated with 0.5 M staurosporine for 12 h before fixation.
Antibodies, Immunofluorescence, and Western Analysis
Antibodies used in this study were rabbit anti-pan-lamin A/C (2032; Cell
Signalling Technology, Danvers, MA), mouse anti-human lamin A/C (SC7292; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-prelamin A
(SC-6214; Santa Cruz Biotechnology), mouse anti-actin (MAB1501R; Millipore
Bioscience Research Reagents, Temecula, CA), and rabbit anti-lamin B1 (SC20682; Santa Cruz Biotechnology).
Immunofluorescence was performed on formaldehyde-fixed cells as described previously (Kudlow et al., 2005). Images were captured on an Axiovert
200 microscope (Carl Zeiss). For Western blotting, cells were lysed in 50 mM
Tris, pH 7.8, with 300 mM NaCl, 50 mM NaF, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 mM dithiothreitol, and protease inhibitors. To resolve
prelamin A from mature lamin A, lysates were run on 8% SDS-PAGE gels.
Westerns were performed as described previously.
RESULTS
Generation of Primary Fibroblasts Expressing Progerin
and Other Mutant Lamin A Proteins
To develop a controlled system in which we could examine
the primary effects of progerin in human fibroblasts, we
developed retroviral vectors to drive stable, high-level expression of lamin A mutants in human cells (Kudlow et al.,
2005). Several lamin A mutants including wild-type lamin
A, progerin (mutant contains 50-amino acid deletion as resulting from G608G mutation), processing-defective L647R
lamin A, and progerin-CS (in which the farnesylation site of
progerin has been mutated to serine) in addition to an empty
vector control were chosen to dissect the function(s) of progerin and phenotypes attributable to stable farnesylation.
AG13353 primary human dermal fibroblasts at PDL 9 were
transduced in parallel with retroviruses driving expression
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B. A. Kudlow et al.
Figure 1. Progerin and L647R lamin A disrupt nuclear morphology in a farnesylation-dependent manner. (A) Western analysis to confirm
expression of each lamin A mutant in AG13353 cells. Progerin runs with characteristic molecular weight between lamin A and lamin C, and
it is detectable in both patient fibroblasts, AG01972C, and progerin-transduced normal dermal fibroblasts. The L647R mutation blocks the
final processing step in lamin A maturation; therefore, this mutant has a molecular weight greater than lamin A and is detectable with
prelamin A antibodies. Progerin-CS fails to undergo any cleavage and is therefore three amino acids longer progerin. The blot was probed
first with lamin A/C antibodies, and then sequentially reprobed for prelamin A and actin. (B) Representative immunofluorescence images
of AG13353 cells transduced with the indicated lamin A mutants. Lamin A/C is stained in green and DNA, stained with DAPI, is in blue.
(C) Percentage of cells showing abnormal nuclear morphology was scored by staining the nuclear periphery with lamin A and the DNA with
DAPI. Cells showing sharp edges, invaginations, or blebs were scored as dysmorphic. Cells were cultured in the indicated concentrations of
FTI R115777 for 48 h. Error bars represent SD of three independent experiments (100 cells per experiment). (D) Verification of the efficacy of
FTI treatment. AG13353 cells were treated with FTI R115777 for 48 h. Cells were lysed and proteins resolved on 8% SDS-PAGE gels. The blots
were probed first with antibodies for lamin A/C, followed by antibodies against prelamin A and actin.
of each of these mutants. The cells were selected for 6 d in
hygromycin-B and, except where indicated, immediately
subjected to further analysis.
Western analysis revealed that each of these mutants was
robustly expressed, with progerin expression in the virally
transduced cells surpassing that of patient fibroblasts (Figure 1A). The L647R mutation results in a farnesylated lamin
A protein that cannot undergo the internal cleavage event
and the resulting protein migrates at a size slightly larger
than mature lamin A. Progerin-CS, which does not undergo
farnesylation and subsequent cleavage of its C-terminal
three amino acids, displays a slightly lower mobility than
progerin.
Patient-derived HGPS fibroblasts are characterized by
dysmorphic nuclei (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). This is suggestive of a gain-of-function
activity of progerin, leading to disruption of the dynamics of
the nuclear lamina. To determine whether virally transduced fibroblasts display the same defective nuclear morphology as patient-derived HGPS fibroblasts, we scored nuclear morphology by staining the DNA with DAPI and the
nuclear periphery with anti-lamin A/C antibodies. As ex5240
pected, progerin, but not wild-type lamin A, had a dramatic
effect on nuclear morphology (Figure 1B). Quantification in
cells transduced with progerin revealed that nearly 80% of
cells had dysmorphic nuclei, compared with only 55% in
patient-derived fibroblasts. To determine the role of farnesylation of progerin in its ability to disrupt nuclear morphology, we examined cells transduced with L647R lamin A and
progerin-CS. Cells expressing L647R lamin A displayed a
similar degree of disrupted nuclear morphology to progerin-expressing cells, whereas cells expressing progerin-CS
seemed normal. Next, we determined whether nuclear morphology in progerin-expressing cells could be restored by
treatment with FTIs as has been reported for patient-derived
HGPS fibroblasts (Capell et al., 2005; Glynn and Glover,
2005; Mallampalli et al., 2005; Toth et al., 2005; Yang et al.,
2005). We find that even at very low concentrations of FTI
(0.05 M R115777), nuclear morphology of the progerin- and
L647R lamin A-transduced cells, like the patient-derived
fibroblasts, was restored to normal (Figure 1C). To determine whether R115777 is effective at these low concentrations, we treated AG13353 cells with the drug and monitored the inhibition of prelamin A processing by Western
Molecular Biology of the Cell
hTERT Suppresses Progerin-linked Defects
Figure 2. Progerin-expressing cells exhibit subtle premature senescence. (A) AG13353 cells transduced with
the indicated lamin A mutants were continuously cultured, and the cumulative population doublings were
monitored until the cells exhibited proliferative arrest.
(B) SA -gal activity was monitored in each culture. The
percentage of SA -gal–positive cells at each time point
is indicated.
analysis. We find that even at concentrations as low as 0.05
M, prelamin A processing is markedly inhibited as evidenced by the accumulation of prelamin A and the depletion
of mature lamin A in these cells. Similar results were obtained with a different set of primary dermal fibroblasts
expressing lamin A mutants (Supplemental Figure 1). Together, these data indicate that the presence of stably farnesylated lamin A is sufficient to alter nuclear morphology.
Furthermore they indicate that prenylation of progerin is
essential for this activity.
Long-Term Culturing of Mutant Lamin A-expressing Cells
Cells derived from HGPS patients, like cells derived from
patients with other progeroid syndromes, have been reported to undergo premature senescence (Huang et al., 2005,
2008). However, in the case of HGPS patient-derived cells,
no isogenic and simultaneously derived control fibroblasts
have been available for direct comparison. We therefore
used our fibroblasts, all produced under identical conditions
and derived simultaneously, to determine whether the proliferative life span of progerin-expressing cells differs from
control fibroblasts. In two independent experiments, using
independently derived fibroblast cultures, we observed that
progerin-expressing fibroblasts underwent growth arrest at
approximately three to four PDLs fewer than control cells
(those expressing wild-type lamin A or empty vector) (Figure 2A). This growth arrest was associated with a marked
increase in senescence-associated -galactosidase (SA -gal)
activity (Figure 2B). Blocking the farnesyl modification of
progerin, using the progerin-CS mutant, seemed to confer a
modest improvement over progerin in that these cells behaved similarly to wild-type lamin A-expressing cells (see
below). This finding indicates that stable farnesylation is
indeed a contributing factor in the premature proliferative
arrest of progerin-expressing cells.
Vol. 19, December 2008
Interestingly, we observed no deleterious effects of expression of L647R lamin A. Instead, expression of stably farnesylated lamin A protein was found to modestly enhance the
proliferative life span of AG13353 fibroblasts. That this mutant differs from progerin in this assay is intriguing; although, we have previously observed distinct activities of
progerin and L647R lamin A, most notably with respect to
retinoblastoma (pRB) stabilization (Nitta et al., 2006). These
differences in the regulation of pRB may partially underlie
the enhanced proliferative life span associated with L647Rlamin A expression (see Discussion).
Finally, cells overexpressing wild-type lamin A, also underwent proliferative arrest at ⬃2 PDLs fewer than the
vector controls (Figure 2A). Cells overexpressing wild-type
lamin A have been reported to accumulate farnesylated
prelamin A, possibly because excess synthesis of prelamin A
saturates the processing machinery (Kudlow et al., 2005).
However, given that cells overexpressing L647R exhibit enhanced rather than diminished proliferative capacity, we
cannot attribute these phenotypes to reduced lamin A processing. Instead, we speculate that excess levels of mature
lamin A have modest inhibitory effects.
Expression of Farnesylated Lamin A Proteins Does Not
Result in Widespread Up-Regulation of p53 Target Genes
Cells derived from HGPS patients have been reported to
display evidence of DNA damage and activation of the p53
pathway (Liu et al., 2005; Scaffidi and Misteli, 2006). To
determine whether p53 target genes are up-regulated in
primary fibroblasts expressing each of the lamin A mutants,
we isolated RNA from each of these cultures and performed
quantitative reverse transcription (qRT)-PCR for p21,
GADD45B, and IGFBP3, each a known p53 target gene. Each
RNA sample was normalized to the geometric mean of three
internal housekeeping control genes: ␥-ACT, TBP, and
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B. A. Kudlow et al.
perhaps in response to infrequent cellular insults that accompany prolonged passage in culture.
Figure 3. Farnesylated lamin A proteins do not induce gross activation of p53 in AG13353 cells. (A) Quantitative RT-PCR was used
to measure mRNA abundance for the p53 target genes P21,
GADD45B, and IGFPBP3. mRNA inputs were normalized to the
combination of three housekeeping genes (see Materials and Methods). For each gene the expression relative to vector controls is
indicated. Error bars represent SD of six independent experiments.
For each gene the p value of each sample compared with the vector
control was ⱖ0.15 except for IGFBP3 where the p value for wildtype lamin A was 0.01 and the p value for L647R lamin A was 0.09
compared with vector controls. (B) Western analysis of p21 protein
levels. Lysates of asynchronous, subconfluent cells were probed
simultaneously for p21 and actin. To induce p21 protein levels, cells
were treated with 5 M camptothecin for 24 h.
GAPDH. Six independent RNA isolations from each culture
were performed. We found no statistically significant difference in the level of P21, GADD45B, or IGFBP3, between cells
expressing any lamin A mutant and the empty vector control cells (Figure 3A). The 1.5-fold increase in IGFBP3 expression in progerin and L647R failed to reach significance
(p ⫽ 0.09). To confirm that we could detect changes in P21
and GADD45B, we treated cells with 5 M camptothecin for
24 h and performed qRT-PCR for these genes. Under these
conditions, we find robust activation of each (Supplemental
Figure 2), indicating that our qRT-PCR is sensitive to
changes in these p53 target genes. Additionally, we saw no
evidence of enhanced P21 expression in progerin-expressing
82-6 fibroblasts (Supplemental Figure 2). Finally, to reconfirm our findings, we measured p21 protein level by western
blot. We found that p21 levels for each of the lamin A
mutant-expressing cultures were similar to those of empty
vector controls (Figure 3B). These data indicate that p53
targets genes are not highly up-regulated in progerin-expressing cells. However, we cannot rule out the possibility
of a low level, transient activation of p53 in these cells
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Immediate Effects of Expression of Farnesylated Lamin A
Proteins
In the course of deriving fibroblasts expressing various
lamin A mutants, we noticed reproducible differences between the transductants in the outgrowth of clones. To
determine the nature of the varied rates of clonal outgrowth,
the cells were plated at clonal density immediately after
selection for transduced cells in hygromycin-B. Clones were
allowed to grow undisturbed for 6 d, at which point the
number of cells per colony was scored. Early passage
AG13353 primary, dermal fibroblasts transduced with a control, empty vector displayed a broad distribution of clone
sizes, with a median clone size of 17 cells (Figure 4A and
Table 1). Only 30% of colonies from these cells contained
four or fewer cells. In contrast, cells transduced with progerin showed a markedly different clone-size distribution.
The median number of cells per clone in progerin-expressing cells was three, with nearly 60% of colonies containing
four or fewer cells. Similarly, expression of a L647R lamin A
produces a marked clonal outgrowth defect, giving rise to a
clone-size distribution skewed toward four-cell and smaller
clones with a median clone size of four cells per clone.
Overexpression of wild-type lamin A or progerin-CS consistently resulted in cells with clonal outgrowth defects of an
intermediate degree with wild-type lamin A cells having a
mean clone size of 8.5 cells and progerin-CS having a mean
clone size of eight cells. The finding that overexpression of
wild-type lamin A can impair proliferation is consistent with
a report by Ivorra et al. (2006) showing that lamin A/C can
interfere with the function of c-Fos, a key mediator of mitogen-stimulated proliferation (Ivorra et al., 2006). In this case,
excess prelamin A resulting from overexpression could be
the culprit because expression of L647R has similar effects.
To further characterize the defect in clonal outgrowth
associated with expression of the various lamin A mutants,
we next assayed the plating efficiency and total cellular
proliferation of each of the cultures. For each culture 100
cells at PDL 1 were plated onto 100-cm plates and allowed to
grow undisturbed for 6 d. At this time, clones were counted
and the total number of cells per plate was determined
(Figure 4B). We found that although the plating efficiency
and clone number for each culture was similar, the total
number of cells that grew out from the 100 initially plated
cells was significantly different between the cultures. Indeed
the vector control cells produced nearly twice as many cells
after 6 d than did the progerin- or L647R-expressing cells.
Similarly, the wild-type lamin A-overexpressing cells and
the progerin-CS-expressing also produced fewer cells than
the vector control, although more than the progerin- and
L647R-expressing cells.
To confirm these findings, we scored the clone-size distribution in 82-6 primary dermal fibroblasts and found similar
results (Supplemental Figure 3). Clonal analysis again revealed that cells stably expressing progerin or L647R had a
significant delay in outgrowth from single cells and that
overexpression of wild-type lamin A had more modest consequences. Together, these findings indicate that permanently farnesylated lamin A variants impair cell proliferation in primary human fibroblasts.
To determine why these cells fail to grow into larger
colonies, we measured the rate of cellular senescence in the
population at early times points. Surprisingly, we found
similar, very low levels of senescence at these time points
(Figure 2B). This suggests that permanent cell cycle exit due
Molecular Biology of the Cell
hTERT Suppresses Progerin-linked Defects
Figure 4. Farnesylated lamin A proteins cause defects in clonal outgrowth. (A) AG13353 cells were transduced with indicated lamin A
mutants and selected for 7 d in hygromycin-B. Immediately after selection, cells were plated at clonal density, allowed to grow for 6 d, at
which point the number of cells per clone was scored (148 clones/sample). (B) 100 cells for each culture at PDL 1 were plated to 10-cm plates.
After 6 d of growth, the total number of clones on the plate was counted to quantify plating efficiency. Similarly, after plating 100 cells per
culture, the total number of cells per plate was counted after 6 d of growth (data are presented as the mean and SD of five independent
experiments). (C) To determine the rate of apoptosis in the cultures, PDL 2 cells were TUNEL stained, and the fraction of TUNEL-positive
nuclei is presented (data represent the mean and SD of 3 independent experiments). (D) The same cultures of AG13353 cells were passaged
for six population doublings before plating at clonal density and scoring clone-size distribution (74 clones/sample).
to senescence does not underlie the failure of cells to form
large colonies. We also noted no significant fraction of apoptotic cells in any of the cultures (Figure 4C). These data lead
us to propose that a subpopulation of the progerin- or
L647R-expressing cells undergoing proliferative delay or
arrest when cultured at very low densities.
We were surprised to find a defect in clonal outgrowth but
not in the overall population doubling time in cells expressing progerin or L647R lamin A. It is possible that this proliferation defect is only manifest at low culture densities.
However, because the maximal clone size for each of the
transductants was the same, it is also possible that a subpopulation of progerin- or L647R-expressing cells rapidly
outgrow the larger proportion of cells that proliferate
slowly. We therefore measured clone-size distribution at
PDL 6, when the cells had been in culture for ⬃2 wk. We
found that the median clone-size had become uniformly
smaller, although on average the progerin- and L647R-expressing remained marginally smaller than all of the other
transductants (Figure 4D). These findings indicate that the
clonal outgrowth phenotype associated with progerin- or
L647R-expression is most pronounced early after expression
and that a subpopulation of cells avoids proliferative complications and ultimately overtakes the population. One possibility, that the faster growing cells do not express lamin A
mutants, is highly unlikely because Western analysis of the
culture after long-term outgrowth indicates that expression
Vol. 19, December 2008
of the lamin A mutant is retained in the faster proliferating
culture (data not shown). It remains possible, however, that
faster proliferating cells in the population have marginally
reduced levels of expression, below some threshold necessary to adversely affect proliferation. An alternative explanation is that the differential behavior of cells expressing
permanently prenylated lamin A mutants results from stochastic events, in which the proliferation of most, but not all,
cells is delayed.
Pathways Altered during Cellular Immortalization
Control Clonal Outgrowth in Progerin-expressing Cells
In the course of developing cell lines expressing progerin
and other lamin A mutants, we noted that immortalized
cells showed no changes in growth rate in response to
expression of any lamin A mutant (data not shown). This
raises the possibility that cellular changes that occur during
immortalization may modulate or alter the effects of progerin and other lamin A-processing mutants.
To address this hypothesis, we made discreet changes in
AG13353 cells that parallel alterations occurring during immortalization. We transduced AG13353 with either an
empty vector (pLXSN) or with the same vector carrying
hTERT, the catalytic subunit of telomerase; human papillomavirus (HPV) E6, to inactivate p53; or HPV E7, to inactivate pRB. Immediately after selection in G418, the cells were
transduced with retroviruses driving expression of the
5243
B. A. Kudlow et al.
Table 1. Summary of clone-size distributions for cells used in this study
Culture
AG13353 ⫹ vector (PDL 1)
AG13353 ⫹ wild-type lamin A (PDL 1)
AG13353 ⫹ Progerin (PDL 1)
AG13353 ⫹ L647R (PDL 1)
AG13353 ⫹ progerin-CS (PDL 1)
AG13353 ⫹ vector (PDL 6)
AG13353 ⫹ wild-type lamin A (PDL 6)
AG13353 ⫹ progerin (PDL 6)
AG13353 ⫹ L647R (PDL 6)
AG13353 ⫹ progerin-CS (PDL 6)
AG13353(pLSXN) ⫹ vector (PDL 1)
AG13353(pLXSN) ⫹ wild-type lamin A (PDL 1)
AG13353(pLXSN) ⫹ progerin (PDL 1)
AG13353(pLXSN) ⫹ L647R (PDL 1)
AG13353(pLSXN-hTERT) ⫹ vector (PDL 1)
AG13353(pLXSN-hTERT) ⫹ wild-type lamin A (PDL 1)
AG13353(pLXSN-hTERT) ⫹ progerin (PDL 1)
AG13353(pLXSN-hTERT) ⫹ L647R (PDL 1)
AG13353(pLSXN-E6) ⫹ vector (PDL 1)
AG13353(pLXSN-E6) ⫹ wild-type lamin A (PDL 1)
AG13353(pLXSN-E6) ⫹ progerin (PDL 1)
AG13353(pLXSN-E6) ⫹ L647R (PDL 1)
AG13353(pLSXN-E7) ⫹ vector (PDL 1)
AG13353(pLXSN-E7) ⫹ wild-type lamin A (PDL 1)
AG13353(pLXSN-E7) ⫹ progerin (PDL 1)
AG13353(pLXSN-E7) ⫹ L647R (PDL 1)
No. of clones Median clone size p value compared with relevant vector control
analyzed
(mean clone size)
(Wilcoxon Rank Sum Test)
148
148
148
148
148
74
74
74
74
74
148
148
148
148
148
148
148
148
148
148
148
148
148
148
148
148
lamin A mutants. Immediately after this second round of
selection, the cells were subjected to the clone-size distribution assay. At this point, cells have not progressed to a point
where proliferative senescence impedes cell growth and division.
The fibroblasts transduced with empty pLXSN displayed
a similar clone-size distribution to the AG13353 parental
cells transduced with the lamin mutants, with the median
clone-size of the progerin- and L647R-expressing cells being
less than half of that of the empty vector control (Figure 5A
and Table 1). In contrast, cells transduced first with pLXSNhTERT followed by the vectors driving expression of lamin
A mutants displayed very similar clone-size distributions
for each lamin A mutant (Figure 5B and Table 1). In this
instance, the median clone size was 13 cells for the vector
control cells, and 17.5 cells, 17 cells, and 15.5 cells for the
cells expressing wild-type lamin A, progerin, and L647R
lamin A, respectively. We made similar observations in 82-6
dermal fibroblasts upon forced expression of hTERT (Supplemental Figure 3). This indicates that hTERT can protect
cells from a proliferative defect associated with progerin
expression, even at time points when senescence in the
culture is negligible.
Similarly, in cells expressing HPV E6, the clone-size distribution of cells expressing each of the lamin A mutants
were similar to those transduced with empty vector (Figure
5C and Table 1). In this instance, all the cultures had a
slightly higher incidence of floating cells, and this was reflected by median clone-size of eight cells, seven cells, seven
cells, and nine cells for empty vector-, wild-type–, progerin-,
and L647R lamin A-transduced cells, respectively. This suggests that p53 may be involved in suppressing clonal outgrowth in progerin- and L647R-expressing cells, although E6
is reported to have p53-independent functions as well
(Klingelhutz et al., 1996; Liu et al., 2007). In contrast, when
AG13353 cells expressing HPV E7 were transduced with the
5244
17.0 (21.5)
8.5 (16.9)
3.0 (13.1)
4.0 (13.0)
8.0 (14.5)
4.0 (6.3)
4.0 (5.7)
3.0 (4.1)
4.0 (5.1)
3.0 (5.8)
14.0 (17.9)
9.0 (14.7)
6.0 (12.9)
5.5 (12.5)
13.0 (19.6)
17.5 (20.7)
17.5 (21.6)
16.0 (21.6)
8.0 (12.8)
7.0 (11.6)
7.0 (11.6)
9.0 (11.8)
15.0 (19.4)
12.0 (16.3)
8.0 (14.0)
7.0 (12.7)
0.12
0.00027
0.00034
0.024
0.96
0.44
0.90
0.83
0.10
0.0020
0.0019
0.96
0.97
0.96
0.81
0.79
0.89
0.39
0.015
0.0017
lamin A mutants and assayed for clone-size distribution, we
found only a marginal restoration of the clone-size distribution for progerin- and L647R-expressing cells (Figure 5D and
Table 1), suggesting that pRB function is not required for the
impaired clonal outgrowth observed in progerin- or L647Rexpressing cells. Western analysis confirmed that progerin
and L647R lamin A were robustly expressed in both the
hTERT- and E6-expressing cells (Figure 6A).
hTERT and HPV E6 Do Not Rescue Nuclear Morphology
hTERT and HPV E6 protect dermal fibroblasts from a defect
in clonal outgrowth associated with progerin and L647R
lamin A expression. To determine whether these interventions block all of the consequences of progerin expression,
we scored nuclear morphology in each of the AG13353derived cultures. In each instance, progerin and L647R lamin
A caused quantitatively and qualitatively similar disruptions of nuclear morphology when expressed, regardless of
the background of the cells in which they were expressed
(Figure 6B). This indicates that hTERT and HPV E6 can
protect cells from the proliferative defect associated with
progerin expression, but they do not suppress abnormalities
in the nuclear architecture caused by progerin expression.
Thus, altered nuclear morphology, although indicative of
altered lamin A function, is not sufficient to cause proliferative defects of cells in culture.
DISCUSSION
Considerable progress has been made in understanding the
molecular defects underlying HGPS by studying phenotypes of patient-derived fibroblasts. Although informative,
these studies suffer two limitations. First, no isogenic nonaffected control cells are available for comparison. Second,
phenotypes observed in patient-derived cells may reflect
adaptation to long-term expression of progerin in vivo or
Molecular Biology of the Cell
hTERT Suppresses Progerin-linked Defects
Figure 5. hTERT and HPV E6 suppress the progerin-associated clonal outgrowth defect. AG13353 cells were first transduced with pLXSN
(A), pLXSN-hTERT (B), pLXSN-E6 (C), or pLXSN-E7 (D) and selected for 10 d in G418. After selection, the cells were then reinfected with
pMXIH vectors carrying the indicated lamin A mutants. Immediately after hygromycin-B selection, the cells were plated at clonal density and
assayed for clone-size distribution (148 clones/sample).
selection for cells capable of growing in culture. Because the
genetics of HGPS strongly argue that HGPS-associated mutations are dominant, neomorphic or hypermorphic alleles
of LMNA, we reasoned that HGPS-like cells could be derived by expression of progerin in a normal primary fibroblast. This system has permitted us to observe both the
immediate effects of progerin expression and the long-term
effects on cells while maintaining isogenic and equivalently
handled control cells. By comparing and contrasting the
results of this study with those using patient-derived fibroblasts, it may be possible to distinguish the direct effects of
progerin expression in cells from the more secondary effects
associated with progerin in the developing organism from
which the cells were derived.
Like patient-derived HGPS fibroblasts, primary dermal
fibroblasts display gross abnormalities in nuclear morphology when transduced with progerin. Similarly, the stably
farnesylated L647R lamin A mutant induces similar morphological changes. Also like patient fibroblasts, the nuclear
morphology irregularities in both of these cultures were
potently rescued by FTI (Capell et al., 2005; Glynn and
Glover, 2005; Mallampalli et al., 2005; Toth et al., 2005; Yang
et al., 2005). Together, these data indicate that stable farnesylation of lamin A is both necessary and sufficient to induce
these defects in nuclear morphology.
The functional significance of the progerin-mediated disruptions to the nuclear lamina is unclear. Recent data indicate that the mechanical properties of the nuclei of progerinexpressing cells are qualitatively different from cells devoid
of lamin A/C, indicating that progerin acts as a gain-offunction allele with respect to its ability to disrupt the nuclear lamina(Dahl et al., 2006). This suggests that progerin
may trigger unique changes to the nuclear lamina and that
these changes could underlie cellular dysfunction associated
Vol. 19, December 2008
with HGPS. However, most, if not all, laminopathies are
associated with altered nuclear morphology and disrupted
dynamics of the nuclear lamina may be a common feature
(Kudlow et al., 2007). How progerin-mediated gross nuclear
abnormalities could lead to a phenotypic outcome different
from those associated with other laminopathy-associated
alleles is unclear. Alternatively, specific subcellular pathways may be uniquely disrupted, either directly or indirectly, by progerin expression; however, the identity of these
pathways remains to be elucidated.
Although proliferative defects have been reported for
HGPS fibroblasts and for fibroblasts from mouse models of
HGPS (Mounkes et al., 2003; Huang et al., 2005; Varela et al.,
2005; Huang et al., 2008), these have yet to be thoroughly
characterized. Because we can produce isogenic cells that are
handled in parallel, we have been able to compare the
proliferative life span of progerin-expressing primary dermal fibroblasts to control cells. We were surprised to find
that although progerin-expressing cells entered proliferative
arrest associated with senescence earlier than control cells,
the difference was relatively subtle. However, we observed a
more evident difference in the proliferative properties when
progerin- or L647R lamin A-expressing cells were grown at
extremely low density immediately after their retroviral
transduction. Under these conditions progerin or L647R expression leads to a deficiency in clonal outgrowth. This
defective proliferation at low culture density is reminiscent
of a decreased wound healing activity of patient-derived
HGPS cells, a process that requires cell migration and proliferation in regions of low cell density (Verstraeten et al.,
2008). At present, the cellular basis for the proliferative
defect has not been determined. We have been able to rule
out both apoptosis and senescence as contributing pathways. Instead, we propose that cells expressing farnesylated
5245
B. A. Kudlow et al.
Figure 6. hTERT and HPV E6 have no effect on nuclear morphology. (A) Western analysis confirming expression of lamin A mutants in each of the pLXSN-transduced AG13353 cultures. (B) Nuclear morphology was scored after staining of the nuclear periphery
with antibodies to lamin A/C and DNA with DAPI. Data represent
the mean ⫾ SD of independent experiments (100 cells/experiment).
lamin A proteins undergo transient or permanent cell cycle
arrest, possibly resulting from a checkpoint-like response.
We took two approaches, measuring p53 activity in bulk
cultures and altering cellular immortalization pathways, to
address the nature of this clonal outgrowth defect, and
arrived at seemingly contradictory results. We first assessed
activation of the p53 pathway by measuring the activity of
three p53 responsive genes. Our findings indicate that there
is not widespread activation of p53 in progerin- or L647Rexpressing cells, although the possibility that small subpopulations of the cells undergo transient activation of p53
in response to some unknown stimulus cannot be ruled out.
Interestingly, studies of HGPS fibroblasts have reported upregulation of p53 target genes (Varela et al., 2005; Scaffidi and
Misteli, 2006). Further experiments will be required to determine whether transient p53 activation could underlie the
clonal outgrowth phenotype we observe upon acute progerin expression. Alternatively, enhanced p53 activity may be
a secondary consequence of in vivo adaptation of fibroblasts
in a progeroid organism.
In a second, functional approach, we found that inactivation of p53 with the HPV E6 oncoprotein rendered normal
fibroblasts unresponsive to progerin or L647R lamin A expression in the clonal outgrowth assay. This finding is consistent with mouse in vivo studies, suggesting that loss of
p53 reduces progeroid phenotypes associated with high levels of farnesylated lamin A (Varela et al., 2005). It is also
noteworthy that neither inactivation of p53 nor expression of
5246
HPV E6, is sufficient to immortalize human fibroblasts (Shay
et al., 1991). Therefore, the suppression of proliferative inhibition by E6 expression is not an indirect consequence of
immortalization.
Similarly, we found a dramatic suppression of the proliferation defect in fibroblasts expressing hTERT. Suppression
of proliferation defects associated with progerin expression
has not been reported previously, although investigators
have found that hTERT expression will immortalize HGPS
fibroblasts (Wallis et al., 2004; Corso et al., 2005; Huang et al.,
2005). Because the suppressive effects of hTERT are apparent
immediately after hTERT expression, we disfavor a model
whereby enhanced telomere length is a primary determinant. Instead, our findings suggest that hTERT can either
block the signals that lead to arrest of clonal outgrowth or
suppress the machinery, which may include the p53 pathway, that implements the cell cycle arrest.
In addition to inhibiting p53, HPV E6 has other activities,
including directly enhancing transcription of hTERT
(Klingelhutz et al., 1996; Gewin and Galloway, 2001). Because hTERT expression is sufficient to suppress the proliferative defects, one possibility is that this activity of E6 can
be ascribed to increased hTERT transcription. However, we
note that E6-stimulated hTERT transcription is reported for
keratinocytes and has not been found in fibroblasts cultures.
Therefore, we do not favor this mechanism. Other activities
of HPV E6 activities have been reported, including regulation of nuclear factor-B signaling (Nees et al., 2001; Munger
and Howley, 2002; James et al., 2006; Tungteakkhun and
Duerksen-Hughes, 2008). Further studies will be required to
assess whether these “noncanonical” functions of E6 play a
role in suppressing the proliferation defects associated with
progerin expression.
The nature of the stimuli that cause the proliferative defect
are unclear. One possibility is that a checkpoint monitoring
nuclear integrity or morphology is activated in HGPS cells.
However, our data suggest that this is not the case, because
in at least two instances we can uncouple changes to nuclear
morphology from changes in proliferation rate. Dermal fibroblasts transduced with hTERT and progerin show dramatic changes in nuclear morphology, but the proliferation
rate of these cells is indistinguishable from control cells. This
suggests that if compromising the dynamics of the nuclear
lamina leads to a checkpoint controlling proliferation, then
this checkpoint is suppressed, directly or indirectly, by
hTERT. Alternatively, overexpression of the catalytic subunit of telomerase may overcome a telomere metabolism
defect that is induced by either direct interference in telomere maintenance by progerin or by the global defects in
nuclear architecture it causes.
Many diseases leading to progeroid phenotypes have
been linked to mutation of genes important for DNA metabolism (Smith et al., 2005). These include xeroderma pigmentosum diseases, Bloom syndrome, and Werner syndrome, which results from inactivation of the WRN DNA
helicase. The mechanisms underlying these diseases remain
to be definitively ascertained but links to repair of DNA
damage and telomere maintenance have been proposed
based on a number of studies. Our findings provide a link
between Werner syndrome and HGPS in that primary cells
either expressing progerin or lacking WRN display proliferative defects that can be rescued by hTERT expression
(Ouellette et al., 2000; Wyllie et al., 2000). Coupled with
murine studies indicating that inactivation of telomerase
can synergize with Wrn loss to promote short life span
and progeroid phenotypes, it is tempting to speculate that
altered telomere dynamics and/or function may be a
Molecular Biology of the Cell
hTERT Suppresses Progerin-linked Defects
common feature of progeroid diseases (Chang et al., 2004;
Du et al., 2004).
The immediate impacts of L647R expression (impaired
proliferation) differ dramatically from the long-term consequences (delayed senescence). These latter effects also differ
from long-term progerin expression, which modestly accelerates senescence. We speculate that differential effects on
pRB stability underlie these differences. A-type lamins are
required for stabilization of pRB (Johnson et al., 2004; Nitta et
al., 2006) and recently we reported that, when expressed in
Lmna⫺/⫺ cells, progerin could function similarly to wildtype lamin A by stabilizing pRB. Not only did L647R expression in Lmna⫺/⫺ cells fail to restore pRB stability, but it
was sufficient to disrupt pRB stability in a dominant manner
when expressed in Lmna⫹/⫹ cells. pRB function is required
for normal entry into senescence, and we propose that the
delayed senescence of L647R-expressing cells is a consequence of pRB inhibition. Because progerin does not share
this function, delayed senescence would not be apparent.
Increasing evidence suggests that A-type lamins act as tumor suppressors in part due to their regulation of the pRB
pathway (Prokocimer et al., 2006; Dorner et al., 2007). Strikingly, an osteosarcoma (often associated with pRB inactivation in humans; Wadayama et al., 1994) was recently detected in an HGPS patient (King et al., 1978; Shalev et al.,
2007). The LMNA mutation in this patient led not to progerin
but rather to expression of an allele containing a smaller
35-amino acid deletion in C-terminus (Shalev et al., 2007). It
will be important to examine the effects of this LMNA mutant on pRB stability. Unlike long-term senescence, pRB
function is unlikely to be important for the impaired proliferation detected immediately after progerin expression because 1) similar effects are seen with L647R expression and
2) coexpression of HPV E6 fails to rescue the defects.
In conclusion, we report proliferate defects associated
with acute, stable progerin expression in primary human
fibroblasts that can be phenocopied by expression of a stably
farnesylated lamin A variant (L647R). The deleterious effects
can be overcome by either p53 inactivation or hTERT expression but not pRB inactivation. These results are consistent with prior studies proposing that expression of farnesylated LMNA alleles lead to p53-dependent checkpoint
response pathways, although we fail to find significant
chronic induction of p53 targets. More surprisingly, hTERT
expression also ameliorates the proliferate defects associated
with progerin expression, leading us to propose that altered
telomere dynamics and/or structure may be an underlying
component of progeroid disease.
ACKNOWLEDGMENTS
We thank Ray Monnat, Peter Rabinovitch, and Vivian McKay for helpful
discussions, as well as Judy Campisi and Denise Galloway for providing
reagents. Michael Gelb kindly provided R115777. B.A.K. was supported in
part by Public Health Service National Research Service Award T32 GM07270
from National Institute of General Medical Sciences. These studies were
supported by National Institutes of Health grant R01 AG-024287 (to B.K.K.)
and by funding from the National Institutes of Health-supported Seattle
Cancer and Aging Program P20 CA-103728.
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Molecular Biology of the Cell