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Genes to Cells (2007) 12, 397-406. doi:10.1111/j.1365-2443.2007.01057.x
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Accumulation of multiple forms of lamin A with down-regulation of FACE-1 suppresses growth in senescent human cells

 Ryo Ukekawa1, Kensuke Miki1, Michihiko Fujii1, Hisashi Hirano2 and Dai Ayusawa1,*

1 Department of Biochemistry, Kihara Institute for Biological Research, Yokohama City University, Maioka-cho 641-12, Yokohama 244-0813, Japan
2 Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Maioka-cho 641-12, Totuka-ku, Yokohama 244-0813, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
5-Bromodeoxyuridine (BrdU) clearly induces a senescence-like phenomenon in every cell type. Proteome analysis revealed that lamin A and C were most highly increased in the nuclei of HeLa cells upon addition of BrdU. Immunoblot analysis also revealed marked accumulation of nuclear prelamin A. Consistently, farnesylated-proteins converting enzyme 1 (FACE-1) was markedly down-regulated in the same cells. Similar phenomena were also observed in normal human fibroblasts undergoing replicative senescence. Immunochemical analysis confirmed the above results. Lamin A is a major component of lamina and responsible for several genetic diseases. Thus, we ectopically expressed a wild-type, a mature type and a premature type of lamin in HeLa cells. All of these forms similarly inhibited colony formation and delayed cell cycle progression mainly through G2 phase. These results suggest that a change in the amount of lamin A, rather than appearance of its truncated form, is responsible for growth retardation in affected cells.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Cellular senescence is originally defined as a terminal phenomenon in mammalian cells in culture (Hayflick 1965). Senescent cells exhibit flat and enlarged cell shape, loss of division potential and induction of specific genes called senescence-associated genes (Dimri et al. 1995). Members of the cyclin-dependent protein kinase inhibitors, p21waf1/sdi-1 and p16ink4a, and the tumor suppressor p53 seem to have a role in arresting growth because they are up-regulated or activated in senescent cells (Nobori et al. 1994). Suboptimal culture conditions and various stresses or damages, which lead to cessation of DNA replication, can also induce phenomena similar to cellular senescence. For instance, hydrogen peroxide, histone deacetylase inhibitors and DNA topoisomerase inhibitors do so in normal human fibroblasts (Ogryzko et al. 1996; Michishita et al. 1998). Since many factors can induce cellular senescence in normal cells, multiple pathways are considered to mediate it. Despite numerous studies, its molecular mechanism is yet to be identified.

5-Bromodeoxyuridine (BrdU) immediately and clearly induces a phenomenon similar to premature senescence in every cell type (Michishita et al. 1999). Historically, BrdU has been used as a modulator of cellular differentiation with cAMP and butyrate (Wilt & Anderson 1972). The latter two are well known to target protein kinase A and histone deacetylase, respectively. We have extensively characterized genes up- or down-regulated by addition of BrdU in various cell lines using PCR-based cDNA subtractive hybridization and DNA micro array analysis (Suzuki et al. 2001a; Minagawa et al. 2004). Such BrdU-responsive genes are found to behave similarly in normal human fibroblasts undergoing replicative senescence. Interestingly, the immediately (12 h) responsive genes are also up- or down-regulated during senescence in normal human fibroblasts, although much higher in late stages of cells BrdU-responsive genes are clustered on particular sites of chromosomes, and seem to have various functions.

On the other hand, BrdU has long been considered to decondense particular regions of chromosomes after incorporation into DNA. In agreement with this, BrdU induces specific genes on particular sites on chromosomes (Suzuki et al. 2001a, 2002; Minagawa et al. 2004, 2005). AT-tract minor groove binders (e.g., distamycin A, Hoechst 33258 and netropsin) and AT-hook proteins (HMG-1 and synthetic multi-AT-hook domains such as MATH2 and MATH20) synergistically stimulate the effect of BrdU to induce senescence markers and genes (Suzuki et al. 2001b; Satou et al. 2004a). 5-Bromouracil-substitution in AT-rich sequences reduces their bending capacity, intensifies their interaction to the nuclear matrix proteins (Ogino et al. 2002) and changes nucleosome assembly on model plasmids in yeast cells (K. Miki et al. manuscript in preparation).

In this study, we undertook a proteome analysis of nuclear proteins in senescent cells to detect a change in chromatin structure. We found that lamin A/C and prelamin A are most dramatically up-regulated in HeLa cells upon addition of BrdU and normal human fibroblasts entering senescence as shown in this study. Lamin A and C are encoded by the LMNA gene by alternative splicing and responsible for various diseases, including Hutchinson–Gilford progeria syndrome (Eriksson et al. 2003; Mounkes et al. 2003). Lamins are highly conserved members of the intermediate filament protein family that constitutes nuclear lamina, a meshwork structure on the nucleoplasmic side of the inner nuclear membrane (Wilson et al. 2001; Hutchison 2002). Mammalian cells have mainly two types of lamins: A-type lamins (A, C, A delta10 and C2) are produced by alternative splicing of the LMNA gene in differentiated cells (Lin & Worman 1993) and B-type lamins (B1 and B2) are expressed from the two distinct genes in nearly all cell types. Mature lamin A (70 kDa) is produced from prelamin A (71 kDa) by multiple post-translational modifications. First, the cysteine of the CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) in the carboxy terminal end is isopreylated, and its AAX terminal tri-peptide is removed by proteolytic cleavage. Second, the cysteine is methylated by isoprenylcysteine carboxyl methyl transferase. Finally, the carboxy terminal 11 residues (amino acid 650–661) are clipped from the protein (Wolda & Glomset 1988). In contrast, lamin C lacks the carboxy terminal CAAX motif and does not receive this processing (Fisher et al. 1986). The prenylation and methylation at the C-terminal cysteine of lamin A are required for its attachment to the inner nuclear membrane (Holtz et al. 1989).

Based on the above findings, we addressed the role of A-type lamins in HeLa cells induced to enter senescence by BrdU and replicative senescent normal human fibroblasts.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Proteome analysis of nuclear proteins

We examined a change in nuclear proteins in HeLa cells upon addition of BrdU employing a very reliable method of 2D-DIGE followed by mass spectrometry. The cells were cultured with BrdU for 96 h since all of the typical senescence-associated genes are induced by 96 h after addition of BrdU (Minagawa et al. 2005). Approximately 1000 protein spots were detected on gels, and nearly 60 spots were found to increase or decrease by more than 1.5-fold under these conditions (Fig. 1). Out of the 60 spots, 40 prominent proteins were identified by peptide mass-fingerprinting analysis (Fig. 1D).


Figure 1
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  		Figure 1  2D-DIGE analysis of nuclear proteins in HeLa cells cultured with or without BrdU. Cy3 and Cy5 labeled protein samples were both loaded on the same gel for 2D analysis. (A) Fluorescence image of Cy3-labeled proteins (red) from control cells. Nuclear proteins were prepared from control cells cultured without BrdU, conjugated with fluorescence dyes and subjected to 2D-DIGE analysis as described in Experimental procedures. (B) Fluorescence image of Cy5-labeled proteins (green) from cells cultured with BrdU. Nuclear proteins were prepared from the cells cultured in the presence of 50 µM BrdU for 4 days and analyzed as in (A). (C) Two-color merged image. Images (A) and (B) were merged to illustrate up-regulated protein spots as green, down-regulated spots as red and unchanged spots as yellow. (D) Identified protein spots. Altered protein spots (arrows) were identified by mass spectrometry as described in Experimental procedures.

 
Among the altered proteins identified, we focused on lamin A (spots 365 and 381) and lamin C (spot 589) because they are among the most abundant nuclear proteins and were most highly (by 4- to 7-fold) increased. The changes in lamin A and C were illustrated by 3-D simulation software (DECYDER). Spot 365 for lamin A was found to increase by 4.6- and 5.4-fold at 48 and 96 h after addition of BrdU, respectively. Similarly, spot 381 for lamin A increased by 7.9- and 7.2-fold, respectively (Fig. 2A). Spot 589 for lamin C increased by 4.6- and 3.9-fold, respectively (Fig. 2B).


Figure 2
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  		Figure 2  Changes in lamin A and C after addition of BrdU in HeLa cells. (A) Three dimensional illustration of lamin A. Samples were prepared from the cells cultured in the presence of 50 µM BrdU for the time indicated, and subjected to 2D-DIGE analysis as in Fig. 1. The upper panels show the region containing spot no. 381 (lamin A). The lower panels show 3D images of the spots created by DECYDER software. Arrows show the peaks of lamin A. (B) Three dimensional illustration of lamin C. Experiments were done as in (A). The upper panels show the region containing spot No. 589 (lamin C). Arrowheads show protein the peaks of lamin C.

 
Blotting analysis of lamin A/C and prelamin A

We examined expression profiles of lamin A and C by immunoblot analysis with specific antibody to lamin A/C (Fig. 3A). Nuclear protein samples were prepared from HeLa cells cultured with BrdU for 96 h. The antibody detected marked increase in both lamin A and C in the nuclei consistent with the results of 2D-DIGE. Interestingly, prelamin A, a precursor of mature lamin A, was found to increase markedly upon addition of BrdU.


Figure 3
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  		Figure 3  Blotting analysis of lamin A/C in HeLa cells cultured with BrdU and in senescent TIG-7 cells. (A) Immunoblot analysis of lamin A/C and prelamin A. Samples of nuclear proteins were prepared from HeLa cells cultured with or without 50 µM BrdU for 4 days and from proliferating (32 PDLs) and senescent (71 PDLs) TIG-7 cells; 10 µg of nuclear proteins were subjected to immunoblotting with specific antibodies to lamin A/C or prelamin A as described in Experimental procedures. The numbers below the lamin A/C and prelamin A blots are the quantification of the signals, normalized to the actin loading control, with the value of the untreated HeLa cells or proliferating cells arbitrarily set as 1. (B) Northern blot analysis of lamin A mRNA. Total RNA samples were prepared from the same cells as in (A) and subjected to Northern blot analysis using probes for lamin A/C or 18 s rRNA as described in Experimental procedures. The signals were quantified and normalized as described in above.

 
The above results led us to examine the behaviors of lamin A/C and prelamin A in normal human fibroblast TIG-7 (Fig. 3A). When cells approached replicative senescence (at 75 PDLs), the protein levels for lamin A and C were not found to significantly change as compared to proliferating TIG-7 cells (at 46 PDLs, a middle stage of passages). However, the expression level for prelamin A was found to markedly increase in senescent TIG-7 cells in sharp contrast to a very low level in proliferating TIG-7 cells. These results demonstrate that prelamin A behaved quite similarly in the two systems of cellular senescence.

Then we examined mRNA levels for lamin A/C by Northern blot analysis. They did not change in HeLa cells upon addition of BrdU or in senescent TIG-7 cells (Fig. 3B). Thus, the up-regulation of lamin A/C and prelamin A is regulated by a post-transcriptional mechanism.

Nuclear localization of lamin A/C

We examined subcellular localization of lamin A/C and prelamin A by staining with rhodamine-conjugated specific antibodies in HeLa and TIG-7 cells. Upon addition of BrdU in HeLa cells, the fluorescence signal for lamin A/C was accumulated mainly in the nuclear rims and slightly in the nucleoplasm within 4 days (Fig. 4A). Alternatively, the signal for prelamin A was accumulated in the nucleoplasm by 4 days after addition of BrdU. In proliferating TIG-7 cells, lamin A/C and prelamin A were localized at the nuclear rims and nucleoplasm similar to untreated HeLa cells (Fig. 4B). When TIG-7 cells approach replicative senescence, the signals for lamin A/C were accumulated mainly on the nuclear rims. Interestingly, the signal for prelamin A seemed to be located in senescent associated heterochromatin foci (SAHF), which are preferentially stained with ligands that bind to AT-rich heterochromatin (Fig. 4B).


Figure 4
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  		Figure 4  Immunofluorescence staining of lamin A/C and prelamin A in HeLa cells cultured with BrdU and normal senescent TIG-7 cells. (A) HeLa cells were cultured in the presence of 50 µM BrdU for 4 days and subjected to indirect immunofluorescence analysis using antibodies to lamin A/C or prelamin A. After staining DNA with 4, 6-diamino-2phenylindole (DAPI), rhodamine fluorescence from a secondary antibody was observed under a fluorescence microscope as described in Experimental procedures. Bars, 15 µm. (B) Immunochemical staining of lamin A/C and prelamin A in proliferating and senescent TIG-7 cells. Proliferating (32 PDLs) and senescent (71 PDLs) cells were subjected to indirect immunofluorescence analysis as described above. Bars, 15 µm.

 
Expression analysis of FACE-1 in senescent cells

We assessed a mechanism by which prelamin A accumulates in senescent cells. As described earlier, the CAAX motif of lamin A is removed by proteolytic cleavage after nuclear import of its precursor form of prelamin A. It is known that the endoprotease, which is termed farnesylated-proteins converting enzyme 1 (FACE-1), a human orthologue of mouse Zmpste 24 and a homologue of yeast metalloproteinase Afc1p/ste24p (Freije et al. 1999; Leung et al. 2001; Goldman et al. 2004), has a major role in processing of prelamin A. We thus examined the mRNA level for this enzyme by Northern blot analysis in senescent cells (Fig. 5). As expected, the level was markedly decreased in HeLa cells within 3 days after addition of BrdU. Similarity, the level was also markedly decreased in TIG-7 cells approaching replicative senescence. These results can well account for the appearance of prelamin A in the two types of senescent cells.


Figure 5
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  		Figure 5  Expression analysis of FACE-1 in senescent cells. Total RNA samples were prepared from HeLa cells cultured with (BrdU) or without (none) 50 µM BrdU for 4 days and from proliferating (38 PDLs) and senescent (73 PDLs) TIG-7 cells, and subjected to Northern blot analysis with specific probe to FACE-1 or human 18 s rRNA as described in Experimental procedures.

 
Ectopic expression of lamin A and its derivatives

To assess a direct role of lamin A in cellular senescence, we ectopically expressed lamin A and its derivatives in HeLa cells and observed their phenotypes. We constructed three plasmids encoding wild-type (wt preLA), a truncated mature form (wt matLA) and a truncated preform (mut preLA) of lamin A each tagged with GFP under the control of CMV promoter. Schematic illustration of lamin A and its derivatives was described in Fig. S1. Mature lamin A is produced by two rounds of proteolytic cleavages of the C-terminal peptides of prelamin A. The C-terminal CAAX motif is involved in the initial proteolytic cleavage and isoprenylation reaction essential for localization onto the nuclear membrane and the second round of proteolytic cleavage (Holtz et al. 1989; Hennekes & Nigg 1994). Thus, the mut preLA having a substitution of 661Cys with Met in the CAAX-motif is unable to produce mature lamin A. The wt matLA has a deletion of C-terminal 15 amino acids and is thus considered to be free from isoprenylation.

The three kinds of plasmids each tagged with GFP were transfected to HeLa cells together with pPGKpuro encoding puromycin N-acetyl transferase. Sub-cellular distribution and expression levels of the ectopic lamins were confirmed by microscopic observation and immunoblot analysis (Fig. 6A,B). Then, transfected cells were selected by addition of puromycin and colonies formed were counted (Fig. 6C). All of the plasmids significantly and similarly reduced colony formation, although the wt preLA was most effective. In these co-transfection experiments, approximately 80% of the colonies selected by puromycin showed GFP fluorescence. Thus, if taking into account the background colonies arisen by unsuccessful co-transfection, all of the plasmids seem to completely inhibit colony formation (Fig. 6D). In another experiment, the same plasmids were transfected to HeLa cells alone and cell cycle distribution of cells was examined 48 h after transfection. In each case, cells in G2/M phase of the cell cycle were increased by approximately 2-fold compared to control cells (Fig. 7). These results demonstrate that transient over-expression of lamin A or its derivatives all delay cell cycle progression mainly though G2/M phase.


Figure 6
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  		Figure 6  Effects of ectopic expression of lamin A and its derivatives on HeLa cells. (A) Location of GFP-tagged lamin A and its derivatives in HeLa cells. Cells were transfected with plasmids encoding GFP-tagged wt preLA, wt matLA or mut preLA. At intervals, the cells were stained with DAPI and examined under a fluorescence microscope. Bars, 20 µm. (B) Expression levels of ectopic GFP-lamins. After transfection of vectors coding GFP-lamins, total cell lysate was prepared from transfectants or control vector (coding GFP) and subjected to immunoblotting as described in Experimental procedures. (C) Effects of GFP-tagged lamins on colony formation. Cells were transfected with expression vectors encoding GFP (control), GFP-tagged wt preLA, wt matLA or mut preLA together with pPGKpuro encoding puromycin N-acetyl transferase (molecular ratios = 3 : 1, respectively). The cells (2 x 105 cells/dish) were seeded on a culture dish and cultured in the presence of 1–2 µg/mL of puromycin for 7 days, and colonies formed were stained with Coomassie Brilliant Blue. (D) Normalized representation of colony suppressing abilities. Colonies were counted as in (C) and expressed relative to control after subtracting the numbers of colonies not expressing GFP. Under the experimental conditions used, co-transfection efficiencies were approximately 80% as determined by coexpression of GFP. Error bars are S.D.

 

Figure 7
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  		Figure 7  Effects of GFP-tagged lamins on cell cycle distribution. Asynchronously growing HeLa cells were transfected with plasmids encoding wt preLA, wt matLA or mut preLA. After culture for 48 h, the cells were harvested and analysed by flow cytometry as described in Experimental procedures.

 
To obtain further information on the roles of lamin A/C in cellular senescence, we examined the effect of down-regulation of lamin A/C. We introduced plasmid encoding siRNA for lamin A/C together with pPGKpuro encoding puromycin N-acetyl transferase to HeLa cells. After selection by puromycin for several weeks, three independent clones expressing siRNA for lamin A/C were isolated and then gene silencing was documented by immunoblotting (Fig. S2A). The protein level was decreased to less than 10% of the control level in all of the clones. To screen for phenotypes, we looked for impaired cell growth and sensitivity to BrdU. Unexpectedly, they grew normally (Fig. S2B left), but they showed higher sensitivity to BrdU than control clones (Fig. S2B right).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Lamin A/C and prelamin A markedly accumulated in the nuclei of HeLa cells induced to a senescent-like state by BrdU and senescent normal human fibroblasts. Since their mRNA levels did not change, we searched for a factor that may influence the accumulation of prelamin A. We were able to find that the mRNA level for FACE-1, which cleaves prelamin A at the CAAX motif, is decreased to one-third of the control level after addition of BrdU in HeLa cells and replicatively senesced normal human fibroblasts. This finding is consistent with the results that FACE-1 knockout mice accumulate prelamin A and show signs of premature senescence (Bergo et al. 2002). Taken together, the accumulation of prelamin A by down-regulation of this enzyme may constitute a novel paradigm for cellular senescence. In addition, the above finding may be very beneficial to establish a model system of progeria syndrome with the use of BrdU.

Anomalous forms of lamin A are well known to give rise to ultrastructural perturbations on the nuclear envelope and functional abnormalities in the nuclei. In Hutchinson–Gilford progeria syndrome, lamin A has an internal deletion (residues 606–656) at the carboxyl terminal region due to abnormal splicing, resulting in loss of the second cleavage site but retaining the isoprenylatable cysteine in the CAAX motif (Goldman et al. 2004). This mutant lamin A behaves like a dominant-negative prelamin A because it causes abnormalities in the nuclear envelope. Lack of a specific metalloproteinase responsible for the maturation of prelamin A also results in progeroid phenotypes in mice and humans. Embryonic fibroblasts with the same defect accumulate DNA damage and chromosome aberrations (Liu et al. 2005). Mice with knockout A-type lamins develop to term with no overt abnormalities, but show severe growth retardation and muscular dystrophy postnatally, and impaired spermatogenesis with oogenesis largely unaffected (Lin & Worman 1993; Alsheimer et al. 2004). Mouse embryonic fibroblasts ectopically expressing an unprocessible form of prelamin A show similar defects in checkpoint response and DNA repair. It is also reported that deletion of the CAAX motif form intranuclear aggregates, and results in the disruption of endogenous lamins A/C in Chinese hamster ovary cells. Taken together, unprocessed prelamin A and truncated lamin A seem to act dominant negatively to perturb lamina structure, DNA damage responses and development in humans and mice. It is also demonstrated that down-regulation of lamin A/C increases the sensitivity to BrdU in HeLa cells. This may imply that disruption of the lamin filament network increases instability of chromatin structure and results in hypersensitivity to BrdU.

Based on the above observations, we moderately over-expressed the wild-type prelaminA, a genetically engineered mature-type lamin A and a mutant prelamin A in HeLa cells. Unexpectedly, all of these forms severely and similarly arrested growth as determined by colony forming assay and cell cycle analysis. These data do not conform to the current scenario that dominant negative mutations can exert growth inhibitory effects. Instead, our results suggest that over- or under-expression of lamin A induces growth arrest irrespective of whether it is normal or mutated. We postulate that an imbalance in nuclear proteins, which must be restrictively controlled under normal conditions, may responsible for the growth arrest. In fact, several lines of evidence support this notion. For example, enforced expression of the ubiquitous nuclear skeletal protein HMG-I, which has diverse roles in chromatin structure and regulation of gene expression (Suzuki et al. 2001b; Satou et al. 2004a), causes growth retardation and sensitizes cells to senescence-inducing stimuli in HeLa cells. Moreover, our hypothesis was also underpinned by findings that normally aged fibroblast and HGPS fibroblasts showed higher percentages of G2/M arrest, lamin A/C suppressed and AP-1 function through reducing c-Fos/c-Jun hetetodimer formation, and high levels of lamin A induced cell cycle arrest in S or G2/M phase (Ivorra et al. 2006).

We considered why a quantitative change in lamin A/C or prelamin A induces cellular senescence. A-type lamins are considered to alter chromatin structure and gene expression by binding to chromatin (Pendas et al. 2002). The lamina maintains extensive interactions with both inner nuclear membrane proteins (INM proteins) and chromatin. At least 18–20 INM proteins (including splice variants) can interact with the lamina. Binding partners of lamin A are the LAP1 family proteins (LAP2{alpha}, emerine and nesprin 1) (Dechat et al. 2000). These INM proteins associate with chromatin through the barrier to autointegration factor (BAF). Therefore, we postulate that disruption of a stoichiometric relationship among molecules interacting with lamin A in the nucleus is a cause of growth arrest.

However, an alternative explanation cannot be ruled out, although it is less likely. LAP2{alpha} binds strongly to pocket C and weakly to pocket B of the retinoblastoma protein RB. Hypophosphorylated RB is anchored in the nucleus by interaction with LAP2{alpha}-lamin A/C complex in G1 of the cell cycle (Markiewicz et al. 2002). Thus, cells lacking lamin A resemble those lacking RB, exhibiting an altered cell-cycle profile and a defect in cell-cycle arrest in response to DNA damage (Johnson et al. 2004). A-type lamins are shown to protect phosphorylated RB from proteasomal degradation. Therefore, accumulation of lamin A/C may stabilize lamin A/C-RB interaction, resulting in delay in cell cycle progression through G1 to S phase of the cell cycle. However, it should be noted that cells are arrested mainly during G2 to M phase upon ectopic expression of lamin A in HeLa cells, in which RB is functionally inactivated.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture

Cervical tumor line HeLa and normal human fibroblast strain TIG-7 were obtained from the Japanese Cancer Research Resources Bank (JCRB). HeLa cells were cultured in ES medium supplemented with 5% fetal calf serum. TIG-7 cells were cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal calf serum. Proliferating TIG-7 cells were used at population doubling levels (PDL) between 32 and 38, and the cells entered replicative senescence at 71–73 PDLs. All the cells were cultured at 37 °C in plastic petri dishes under 5% CO2 and 95% humidity.

2-D fluorescence difference gel electrophoresis (2D-DIGE)

Nuclei were prepared from cells as previously described (Satou et al. 2004b). Extraction of nuclei was done as described by Jung et al. (2000). Nuclei were sonicated and digested with Benzonase (Merk) and solubilized in lysis buffer [30 mM Tris–HCl (pH 8.5), 7 M urea, 2 M thiourea, and 4% (w/v) CHAPS]. After solubilization, the samples were centrifuged at 40 000 g for 1 h to remove insoluble material and desalted by centrifugation at 7500 g for 30–60 min through a filter (Amicon Ultra, Millipore). Protein concentrations were determined with a Bio-Rad Protein Assay Kit (Bio-Rad).

The samples were labeled with N-hydroxy succinimidyl ester-derivatives of cyanine dyes Cy2, Cy3 and Cy5 (GE Healthcare Biosciences) according to the protocol previously described (Tonge et al. 2001). After labeling, these samples were loaded on precast immobilized pH gradient strips (24-cm pH 3–10 NL, GE Healthcare Biosciences) set in an Ettan IPGphor isoelectric focusing system (GE Healthcare Biosciences) and subjected to isoelectric focusing for a total of 90 000 V/h (hold at 500 V for 500 V/h, hold at 1 kV for 1 kV/h and hold at 6 kV for 90 kV/h). The strips were transferred on the tops of 20 x 24 cm 12.5% SDS-polyacrylamide gels and electrophoresed with Ettan DALTtwelve apparatus (GE Healthcare Biosciences). After electrophoresis, the gels were scanned with TYPHOON 9400 and analyzed with DECYDER software (GE Healthcare Biosciences).

Identification of protein by mass spectrometry

Protein spots were excised and in-gel digested with trypsin. The resulting peptides were dried in vacuo, dissolved in HPLC buffer A [water: formic acid: TFA; 99.89: 0.1: 0.01] and subjected to HPLC-MS/MS analysis in a Q-Tof Ultima (Micromass) machine connecting by on-line a capillary liquid chromatography (Waters). Data were obtained and analyzed with MASSLYNX and BIOLYNX (Micromass).

Indirect immunofluorescence staining

Cells grown on a glass coverslip were fixed in ice-cold methanol or paraformaldehyde at room temperature for 15 min and immediately washed with PBS for 30 min. The cells were permeabilized with 0.1% Triton-X/PBS at room temperature for 5 min, incubated in 1% bovine serum albumin in PBS for 1 h and reacted with goat polyclonal antibodies against lamin A/C or and prelamin A (sc-6215 and sc-6214, Santa Cruz Biotechnology) at 4 °C over night. The primary antibodies were detected by rhodamine-conjugated chicken anti-goat IgG (sc-2860, Santa Cruz Biotechnology). Photographs were taken under a fluorescence microscope controlled by an IPLab system.

Vector construction

Human lamin A cDNA (wt preLA) was amplified from total RNA prepared from HeLa cells by RT-PCR with the following primers, including the initiation and termination codons, respectively: 5'-AATTGATATCATGGAGACCCCGTCCCA-3' (forward) and 5'-GGTCGGATATCTTACATGATGCTGCAGTTC-3' (reverse). The amplified sequence was cloned into pGEM-T Easy vector (Promega, UK) followed by sequencing. A truncated prelamin A (mut preLA) and truncated mature lamin A (wt matLA) were made by using site-directed mutagenesis (Imai et al. 1991). After confirming the sequences, plasmids encoding lamin A and its mutant forms tagged with green fluorescence protein (GFP-wt preLA, GFP-wt matLA, and GFP-mut preLA) were made as follows. GFP sequence was cloned into the HindIII site of pBluescript II SK (-). Lamin A fragments digested by EcoRV were cloned into the site of pBluescript II SK(-) containing GFP sequence at the EcoRVsite. These plasmids were digested with XhoI and NotI site; the resulting fragments were cloned into the XhoI and NotI site of pDsRed-N1 (Clontech) from which the DsRed sequence was removed.

Construction of recombinant pSilencer expressing siRNA for lamin A/C

Plasmid pSilencer1.0-U6 (Ambion Inc., Austin, TX, USA) was used for DNA vector-based siRNA synthesis under the control of U6 promoter in vivo. The accession numbers given below in brackets are from GENBANK. The siRNA sequence targeting lamin A/C (NM_005572 [GenBank] ) was from position 608–630 relative to the start codon. The sequences of the oligonucleotides used were forward, 5'-AACTGGACTTCCAGAAGAACATTCAAGAGATGTTCTTCTGGAAGTCCAGTTTTTTTT-3' and reverse, 5'-AATTAAAAAAAACTGGACTTCCAGAAGAACATCTCTTGAATGTTCTTCTGGAAGTCCAGTTGGCC-3'. The oligonucleotides duplex formation (annealing) was performed as previously described (Hossain et al. 2006).

Growth and cell cycle analysis

Plasmid encoding lamin A or its derivatives were electroporated into cells under the conditions of 90 V and 20 m s (BEX electroporater, Tokyo, Japan) with plasmid pPGKpuro containing the puromycin resistant gene. For colony formation assays, HeLa cells were transfected with plasmids and cultured with puromycin for 7–10 days. The number of colonies was counted after staining with Coomassie Brilliant Blue.

To examine the effects of plasmids on cell cycle, transfected cells were collected 48 h after transfection, washed with PBS and incubated with 0.5 mg/mL RNase A for 30 min. After staining with 0.05 mg/mL propidium iodide for 15 min, the cells were analysed in a flow cytometer (Beckman).

Immunoblot analysis

Protein lysates (10 µg per lane) were separated on 8% and 12.5% SDS-polyacrylamide gels and blotted on to nitrocellulose membrane. Blots were blocked with 5% (w/v) skim milk in 1% Tween 20 in PBS and incubated with first antibodies (lamin A/C, prelamin A and actin, Santa Cruz, lamin B1, Matritech) with constant shaking. After three washes with the Tween solution, the membrane was incubated with rabbit anti-goat antibody conjugated with horseradish peroxidase. The second antibody was detected with the ECL Western Blotting Detection system (GE Healthcare Biosciences) on X-ray films. Nuclear lysates were subjected to standard immunoblotting as previously described (Satou et al. 2004b). Lamin B1 antibody (monoclonal 101-B7, Matritech) was kindly provided by Dr T. Haraguchi.

Northern blot analysis

Total RNA samples were prepared from cells using an Isogen kit (Nippon Gene, Japan) according to the manufacturer's protocol. The samples (10 µg per lane) were subjected to electrophoresis through 1% formaldehyde agarose gel and transferred onto a nylon membrane followed by cross-linking with ultraviolet light. The membrane was hybridized with 32P-labeled probes for lamin A/C or 18 s rRNA and washed as previously described (Michishita et al. 1999). The membrane was subjected to autoradiography and a quantitative analysis using an image analyzer FLA-5000 (Fuji Photo Film).


   Acknowledgements
 
We thank Dr Tokuko Haraguchi and Takako Koujin (Kansai Advanced Research Center) for introducing us to immunofluorescence staining technology, providing lamin B1 antibody and the valuable discussions, Drs Joe Hirano and Yuki Ishida (Amersham Biosciences) for DeCyeder training and technical advice.


   Footnotes
 
Communicated by: Shunsuke Ishii

* Correspondence: E-mail: dayusawa{at}yokohama-cu.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 24 August 2006
Accepted: 18 December 2006




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