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Vol. 19, Issue 12, 5238-5248, December 2008
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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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 reminiscent of premature aging. The most common LMNA-associated 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).
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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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 µg/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 µg/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 µg/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 4,6-diamidino-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."
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 β-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-β-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 β-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 µM 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 (SC-7292; 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 (SC-20682; 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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 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.
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; 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 expected, 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 µM 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 analysis. We find that even at concentrations as low as 0.05 µM, 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 β-galactosidase (SA β-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.
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). These differences in the regulation of pRB may partially underlie the enhanced proliferative life span associated with L647R-lamin 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 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 µM 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 perhaps in response to infrequent cellular insults that accompany prolonged passage in culture.
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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.
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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 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 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 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 pLXSN-hTERT 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.
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; Liu et al., 2007). In contrast, when AG13353 cells expressing HPV E7 were transduced with the 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 L647R-expressing cells. Western analysis confirmed that progerin and L647R lamin A were robustly expressed in both the hTERT- and E6-expressing cells (Figure 6A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 progerin-expressing cells are qualitatively different from cells devoid of lamin A/C, indicating that progerin acts as a gain-of-function 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 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 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 L647R-expressing 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 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 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 wild-type 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.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347. 
Address correspondence to: Brian K. Kennedy (bkenn{at}u.washington.edu)
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cao, H., and Hegele, R. A. (2003). LMNA is mutated in Hutchinson-Gilford progeria (MIM 176670) but not in Wiedemann-Rautenstrauch progeroid syndrome (MIM 264090). J. Hum. Genet 48, 271–274.[CrossRef][Medline]
Capell, B. C., Erdos, M. R., Madigan, J. P., Fiordalisi, J. J., Varga, R., Conneely, K. N., Gordon, L. B., Der, C. J., Cox, A. D., and Collins, F. S. (2005). Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome. Proc. Natl. Acad. Sci. USA 102, 12879–12884.
Chang, S., Multani, A. S., Cabrera, N. G., Naylor, M. L., Laud, P., Lombard, D., Pathak, S., Guarente, L., and DePinho, R. A. (2004). Essential role of limiting telomeres in the pathogenesis of Werner syndrome. Nat. Genet 36, 877–882.[CrossRef][Medline]
Corso, C., Parry, E. M., Faragher, R. G., Seager, A., Green, M. H., and Parry, J. M. (2005). Molecular cytogenetic insights into the ageing syndrome Hutchinson-Gilford Progeria (HGPS). Cytogenet. Genome Res 111, 27–33.[CrossRef][Medline]
Dahl, K. N., Scaffidi, P., Islam, M. F., Yodh, A. G., Wilson, K. L., and Misteli, T. (2006). Distinct structural and mechanical properties of the nuclear lamina in Hutchinson-Gilford progeria syndrome. Proc. Natl. Acad. Sci. USA 103, 10271–10276.
De Sandre-Giovannoli, A. et al. (2003). Lamin A truncation in Hutchison-Gilford progeria. Science 300, 2055.
Dechat, T., Pfleghaar, K., Sengupta, K., Shimi, T., Shumaker, D. K., Solimando, L., and Goldman, R. D. (2008). Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev 22, 832–853.
Dimri, G. P. et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363–9367.
Dorner, D., Gotzmann, J., and Foisner, R. (2007). Nucleoplasmic lamins and their interaction partners, LAP2alpha, Rb, and BAF, in transcriptional regulation. FEBS J 274, 1362–1373.[CrossRef][Medline]
Du, X. et al. (2004). Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes. Mol. Cell. Biol 24, 8437–8446.
Eriksson, M. et al. (2003). Recurrent de novo point mutations in lamin A cause Hutchison-Gilford progeria syndrome. Nature 423, 293–298.[CrossRef][Medline]
Fong, L. G., Frost, D., Meta, M., Qiao, X., Yang, S. H., Coffinier, C., and Young, S. G. (2006). A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria. Science 311, 1621–1623.
Frock, R. L., Kudlow, B. A., Evans, A. M., Jameson, S. A., Hauschka, S. D., and Kennedy, B. K. (2006). Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation. Genes Dev 20, 486–500.
Gewin, L., and Galloway, D. A. (2001). E box-dependent activation of telomerase by human papillomavirus type 16 E6 does not require induction of c-myc. J. Virol 75, 7198–7201.
Glynn, M. W., and Glover, T. W. (2005). Incomplete processing of mutant lamin A in Hutchinson-Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition. Hum. Mol. Genet 14, 2959–2969.
Huang, S., Chen, L., Libina, N., Janes, J., Martin, G. M., Campisi, J., and Oshima, J. (2005). Correction of cellular phenotypes of Hutchinson-Gilford Progeria cells by RNA interference. Hum. Genet 118, 444–450.[CrossRef][Medline]
Huang, S., Risques, R. A., Martin, G. M., Rabinovitch, P. S., and Oshima, J. (2008). Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A. Exp. Cell Res 314, 82–91.[CrossRef][Medline]
Ivorra, C., Kubicek, M., Gonzalez, J. M., Sanz-Gonzalez, S. M., Alvarez-Barrientos, A., O'Connor, J. E., Burke, B., and Andres, V. (2006). A mechanism of AP-1 suppression through interaction of c-Fos with lamin A/C. Genes Dev 20, 307–320.
James, M. A., Lee, J. H., and Klingelhutz, A. J. (2006). Human papillomavirus type 16 E6 activates NF-kappaB, induces cIAP-2 expression, and protects against apoptosis in a PDZ binding motif-dependent manner. J. Virol 80, 5301–5307.
Johnson, B. R., Nitta, R. T., Frock, R. L., Mounkes, L., Barbie, D. A., Stewart, C. L., Harlow, E., and Kennedy, B. K. (2004). A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation. Proc. Natl. Acad. Sci. USA 101, 9677–9682.
King, C. R., Lemmer, J., Campbell, J. R., and Atkins, A. R. (1978). Osteosarcoma in a patient with Hutchinson-Gilford progeria. J. Med. Genet 15, 481–484.
Klingelhutz, A. J., Foster, S. A., and McDougall, J. K. (1996). Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 380, 79–82.[CrossRef][Medline]
Kudlow, B. A., Jameson, S. A., and Kennedy, B. K. (2005). HIV protease inhibitors block adipocyte differentiation independently of lamin A/C. AIDS 19, 1565–1573.[Medline]
Kudlow, B. A., Kennedy, B. K., and Monnat, R. J., Jr. (2007). Werner syndrome and Hutchinson-Gilford progeria: mechanistic basis of human progeroid syndromes. Nat. Rev. Mol. Cell Biol 8, 394–404.[CrossRef][Medline]
Lammerding, J., Fong, L. G., Ji, J. Y., Reue, K., Stewart, C. L., Young, S. G., and Lee, R. T. (2006). Lamins A and C but not lamin B1 regulate nuclear mechanics. J. Biol. Chem 281, 25768–25780.
Lammerding, J., Schulze, P. C., Takahashi, T., Kozlov, S., Sullivan, T., Kamm, R. D., Stewart, C. L., and Lee, R. T. (2004). Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. J. Clin. Invest 113, 370–378.[CrossRef][Medline]
Liu, B. et al. (2005). Genomic instability in laminopathy-based premature aging. Nat. Med 11, 780–785.[CrossRef][Medline]
Liu, Y., Heilman, S. A., Illanes, D., Sluder, G., and Chen, J. J. (2007). p53-independent abrogation of a postmitotic checkpoint contributes to human papillomavirus E6-induced polyploidy. Cancer Res 67, 2603–2610.
Lutz, R. J., Trujillo, M. A., Denham, K. S., Wenger, L., and Sinensky, M. (1992). Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina. Proc. Natl. Acad. Sci. USA 89, 3000–3004.
Mallampalli, M. P., Huyer, G., Bendale, P., Gelb, M. H., and Michaelis, S. (2005). Inhibiting farnesylation reverses the nuclear morphology defect in a HeLa cell model for Hutchinson-Gilford progeria syndrome. Proc. Natl. Acad. Sci. USA 102, 14416–14421.
McKeon, F. D., Kirschner, M. W., and Caput, D. (1986). Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature 319, 463–468.[CrossRef][Medline]
Mounkes, L. C., Kozlov, S., Hernandez, L., Sullivan, T., and Stewart, C. L. (2003). A progeroid syndrome in mice caused by defects in A-type lamins. Nature 423, 298–301.[CrossRef][Medline]
Munger, K., and Howley, P. M. (2002). Human papillomavirus immortalization and transformation functions. Virus Res 89, 213–228.[CrossRef][Medline]
Nees, M., Geoghegan, J. M., Hyman, T., Frank, S., Miller, L., and Woodworth, C. D. (2001). Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes. J. Virol 75, 4283–4296.
Nitta, R. T., Jameson, S. A., Kudlow, B. A., Conlan, L. A., and Kennedy, B. K. (2006). Stabilization of the retinoblastoma protein by A-type nuclear lamins is required for INK4A-mediated cell cycle arrest. Mol. Cell. Biol 26, 5360–5372.
Nitta, R. T., Smith, C. L., and Kennedy, B. K. (2007). Evidence that proteasome-dependent degradation of the retinoblastoma protein in cells lacking A-type lamins occurs independently of gankyrin and MDM2. PLoS ONE 2, e963.
Ouellette, M. M., McDaniel, L. D., Wright, W. E., Shay, J. W., and Schultz, R. A. (2000). The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes. Hum. Mol. Genet 9, 403–411.
Prokocimer, M., Margalit, A., and Gruenbaum, Y. (2006). The nuclear lamina and its proposed roles in tumorigenesis: projection on the hematologic malignancies and future targeted therapy. J. Struct. Biol 155, 351–360.[CrossRef][Medline]
Rober, R. A., Weber, K., and Osborn, M. (1989). Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study. Development 105, 365–378.[Abstract]
Rubio, M. A., Kim, S. H., and Campisi, J. (2002). Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells. J. Biol. Chem 277, 28609–28617.
Scaffidi, P., and Misteli, T. (2005). Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Nat. Med 11, 440–445.[CrossRef][Medline]
Scaffidi, P., and Misteli, T. (2006). Lamin A-dependent nuclear defects in human aging. Science 312, 1059–1063.
Scaffidi, P., and Misteli, T. (2008). Lamin A-dependent misregulation of adult stem cells associated with accelerated ageing. Nat. Cell Biol 10, 452–459.[CrossRef][Medline]
Shalev, S. A., De Sandre-Giovannoli, A., Shani, A. A., and Levy, N. (2007). An association of Hutchinson-Gilford progeria and malignancy. Am J. Med. Genet A 143A, 1821–1826.
Shay, J. W., Pereira-Smith, O. M., and Wright, W. E. (1991). A role for both RB and p53 in the regulation of human cellular senescence. Exp. Cell Res 196, 33–39.[CrossRef][Medline]
Sinensky, M., Fantle, K., Trujillo, M., McLain, T., Kupfer, A., and Dalton, M. (1994). The processing pathway of prelamin A. J. Cell Sci 107, 61–67.[Abstract]
Smith, E. D., Kudlow, B. A., Frock, R. L., and Kennedy, B. K. (2005). A-type nuclear lamins, progerias and other degenerative disorders. Mech. Ageing Dev 126, 447–460.[CrossRef][Medline]
Spann, T. P., Goldman, A. E., Wang, C., Huang, S., and Goldman, R. D. (2002). Alteration of nuclear lamin organization inhibits RNA polymerase II-dependent transcription. J. Cell Biol 156, 603–608.
Spann, T. P., Moir, R. D., Goldman, A. E., Stick, R., and Goldman, R. D. (1997). Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis. J. Cell Biol 136, 1201–1212.
Toth, J. I., Yang, S. H., Qiao, X., Beigneux, A. P., Gelb, M. H., Moulson, C. L., Miner, J. H., Young, S. G., and Fong, L. G. (2005). Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes. Proc. Natl. Acad. Sci. USA 102, 12873–12878.
Tungteakkhun, S. S., and Duerksen-Hughes, P. J. (2008). Cellular binding partners of the human papillomavirus E6 protein. Arch. Virol 153, 397–408.[CrossRef][Medline]
Varela, I. et al. (2005). Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53 signalling activation. Nature 437, 564–568.[CrossRef][Medline]
Verstraeten, V. L., Ji, J. Y., Cummings, K. S., Lee, R. T., and Lammerding, J. (2008). Increased mechanosensitivity and nuclear stiffness in Hutchinson-Gilford progeria cells: effects of farnesyltransferase inhibitors. Aging Cell 7, 383–393.[CrossRef][Medline]
Wadayama, B., Toguchida, J., Shimizu, T., Ishizaki, K., Sasaki, M. S., Kotoura, Y., and Yamamuro, T. (1994). Mutation spectrum of the retinoblastoma gene in osteosarcomas. Cancer Res 54, 3042–3048.
Wallis, C. V., Sheerin, A. N., Green, M. H., Jones, C. J., Kipling, D., and Faragher, R. G. (2004). Fibroblast clones from patients with Hutchinson-Gilford progeria can senesce despite the presence of telomerase. Exp. Gerontol 39, 461–467.[CrossRef][Medline]
Wyllie, F. S., Jones, C. J., Skinner, J. W., Haughton, M. F., Wallis, C., Wynford-Thomas, D., Faragher, R. G., and Kipling, D. (2000). Telomerase prevents the accelerated cell ageing of Werner syndrome fibroblasts. Nat. Genet 24, 16–17.[CrossRef][Medline]
Yang, S. H. et al. (2005). Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation. Proc. Natl. Acad. Sci. USA 102, 10291–10296.
Yang, S. H., Meta, M., Qiao, X., Frost, D., Bauch, J., Coffinier, C., Majumdar, S., Bergo, M. O., Young, S. G., and Fong, L. G. (2006). A farnesyltransferase inhibitor improves disease phenotypes in mice with a Hutchinson-Gilford progeria syndrome mutation. J. Clin. Invest 116, 2115–2121.[CrossRef][Medline]
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0.15 except for IGFBP3 where the p value for wild-type 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 µM camptothecin for 24 h.
