Materials and Methods

Cell culture

Myofibroblasts were isolated from a granulation tissue pouch in the rat dorsal subcutaneous tissue as described previously (^{Goto et al, 1998}). Briefly, the granulation tissue pouch was created in male Wister rats (120–150 g) by the injection of 30 ml of air into the dorsal subcutaneous tissue followed by 1 ml of 0.1% croton-oil dissolved in maize oil (^{Appleton et al, 1992}). After 23–26 d, the pouch was dissected and digested using 2000 U per ml dispase (Godo-Shusei, Japan) at 37°C for 1 h with gentle stirring. The myofibroblasts obtained were maintained in Dulbecco' modified Eagle' medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 100 U per ml penicillin G (Meiji-Seika, Japan), and 100 g per ml streptomycin (Meiji-Seika). Cells in passages 2–8 were used for all experiments. To isolate and culture normal fibroblasts, connective tissue was dissected from the dorsal subcutaneous region of normal Wister rats and treated in the same way as described above.

Induction of apoptosis

Myofibroblasts were plated on plastic tissue culture dishes or collagen-coated coverslips. After 24 h incubation, cells were treated with STS (0–1000 nM, Wako, Japan), cisplatin (1 mM, Sigma), bleomycin (100 ng per ml, Wako), anti-Fas monoclonal antibody (clone CH-11, 500 ng per ml, MBL, Japan), tumor necrosis factor (TNF-) (50 ng per ml, Genzyme TECHNE, Minneapolis, MN), interferon- (IFN-) (1000 U per ml, Genzyme TECHNE), transforming growth factor 1 (TGF-) (100 ng per ml, King Brewing, Japan), platelet-derived growth factor BB (PDGF-BB) (100 ng per ml, PeproTech, U.K.), or endothelin-1 (1 M, Peptide Institute, Japan) for the indicated periods.

Flow cytometry analysis

Apoptotic cells were quantified using flow cytometry. Myofibroblasts were incubated with or without stimulants for the indicated periods and then harvested by trypsin/ethylenediamine tetraacetic acid (Gibco BRL) treatment. The cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) using a MEBCYTO Apoptosis Kit (MBL). Cells 110⁴ for each sample were analyzed with a Becton-Dickinson FACScalibur (Franklin Lakes, NJ).

Fluorescence staining

Myofibroblasts plated on collagen-coated coverslips were washed with phosphate-buffered saline. For the assessment of nuclear morphology, cells were stained with 1 M of Hoechst 33342 dye (Molecular Probes, Eugene, OR) and observed using fluorescence microscopy (Olympus, Japan). Immunostaining of -SM actin was performed as described previously with slight modifications (^{Goto et al, 1998}). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% (vol/vol) Triton-X 100 in phosphate-buffered saline (Sigma). After blocking with 5% skimmed milk for 30 min, fixed cells on coverslips were probed with a mouse monoclonal anti--SM actin antibody (1:100 dilution, Progen, Germany) for 1 h at room temperature followed by incubation with rabbit antimouse IgG+IgA+IgM-biotin (Nichirei, Japan) for 30 min at room temperature. Cells were then incubated with streptavidin-FITC conjugate (1:50 dilution, Gibco BRL) for 30 min and washed with phosphate-buffered saline. The cells were observed with fluorescence microscopy.

Immunoblotting

Samples were separated by 12% (for -SM actin, all actin isoforms, -actin) or 15% (for caspase-3) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane using a semidry transfer system (1 h, 15 V). After blocking with 5% skimmed milk for 30 min, the membrane was probed with a monoclonal anti--SM actin antibody, a monoclonal antibody to all actin isoforms (clone C4, Chemicon, Temecula, CA), a monoclonal anti--actin antibody (clone AC-74, Sigma), or a polyclonal anticaspase-3 antibody (Upstate Biotechnology, Lake Placid, NY) for 1 h at room temperature. The membrane was washed three times and incubated with horseradish-peroxidase-conjugated antimouse IgG or horseradish-peroxidase-conjugated goat antirabbit IgG (Bio-Rad, Hercules, CA) for 30 min. Immunoreactive proteins were then visualized by treatment with a detection reagent (Super Signal West Dura, Pierce, Rockford, IL). An optical densitometric scan was performed using Science Lab99 Image Gauge Software (Fujiphoto Film, Japan).

Caspase-3 activity assay

Myofibroblasts (110⁶ cells) were incubated with or without stimulants for the indicated periods and lysed on ice for 10 min. The cell lysate (100 l) was incubated with 200 M DEVD-p-nitroanilide (CPP/Caspase-3 colorimetric protease assay kit, MBL) at 37°C for 3 h. Fluorescence of the samples was measured at 405 nm to determine the caspase-3 activity.

Purification of -SM actin

-SM actin was purified from an acetone-dried powder of freshly isolated bovine aorta according to the procedure of^{Spudich and Watt (1971)}. The acetone-dried powder (1 g) was extracted with 20 ml of buffer A (2 mM Tris-HCl pH 8.0, 0.2 mM CaCl₂, 0.5 mM -mercaptoethanol, and 0.2 mM ATP) for 30 min at 0°C and filtered through a coarse sintered glass funnel. The residue was washed with 100 ml of buffer A. The flow through was centrifuged at 10,000g for 1 h at 2°C. Three molar KCl solution was added to the supernatant to give a final concentration of 50 mM and the actin was allowed to polymerize for 2 h at 25°C. Three molar KCl solution was then added to give a final concentration of 0.6 M and the sample was stirred gently for 1.5 h at room temperature. The solution was centrifuged at 80,000g for 3 h at 25°C and the pellet was resuspended with 3 ml of buffer A. The sample was dialyzed against buffer A with vigorous stirring for 3 d changing the buffer every 24 h. The G-actin was obtained by centrifugation at 80,000g for 3 h at 2°C and polymerized in 50 mM KCl.

Immunoprecipitation of caspase-3

Myofibroblasts were incubated with or without STS and washed with phosphate-buffered saline. Cells were lysed on ice for 10 min and insoluble cell debris was removed by centrifugation at 5000 rpm for 3 min. The cell lysate was precleared with protein A-sepharose CL-4B (Sigma) and then incubated with an anticaspase-3 antibody (4.5 g) and protein A-sepharose CL-4B (5 mg) at 4°C for 2 h in 1 ml of the ICE assay buffer [20 mM HEPES pH 7.5, 2 mM dithiothreitol, 10% (vol/vol) glycerol]^{Mashima et al, 1997}) supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 g per ml leupeptin, and 1 g per ml pepstatin. After incubation, the protein bound with the antibody/protein A-sepharose complex was precipitated by centrifugation at 15,000 rpm for 5 min and washed three times with the ICE assay buffer. The immunoprecipitated caspase-3 was used for the -SM actin cleavage assay.

-SM actin cleavage assay in vitro

Bovine aortic smooth muscle actin was labeled with biotin-(AC5)₂ Sulfo-Osu (Biotinylation Kit, Dojindo Laboratory, Japan). The biotinylated actin (0.4 g) was incubated with immunoprecipitated caspase-3 or 12 units of human recombinant active caspase-3/CPP32 (MBL) for 2 h at 37°C in 50 l of ICE assay buffer. The samples were separated by 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk for 30 min, the membrane was incubated with horseradish-peroxidase-conjugated avidin (Bio-Rad) for 1 h at room temperature. The membrane was washed three times and treated with detection reagent (Super Signal West Dura).

Results

Characterization of the cells isolated from the granulation tissue pouch

Myofibroblasts are distinguished from fibroblasts by the expression of -SM actin, a contractile protein that provides myofibroblasts with strong contractility (^{Serini and Gabbiani, 1999}). We performed immunostaining and immunoblotting for -SM actin to confirm that the cultured cells isolated from the granulation tissue pouch were myofibroblasts. As shown in Figure 1a, the majority of the cells exhibited prominent bundles of -SM actin, running parallel to the longitudinal axis of the cells. We previously reported that approximately 90% of the cells obtained from the granulation tissue pouch showed prominent bundles of -SM actin (^{Goto et al, 1998}). A Western blot analysis showed that both the granulation tissue pouch and the cultured cells from the pouch were rich in -SM actin, whereas the fibroblasts from the normal dorsal subcutaneous tissue did not express -SM actin (Figure 1b). These results indicated that the cultured cells isolated from the granulation tissue pouch were mainly myofibroblasts.

Figure 1.

Characterization of the cells isolated and cultured from the granulation tissue pouch. (A) Immunofluorescence staining with anti--SM actin antibody. Cells were plated and grown on collagen-coated coverslips and fixed with 4% paraformaldehyde. Samples were probed with anti--SM actin antibody. Scale bar: 10 m. (B) Western blot analysis with anti--SM actin antibody. Samples were separated by 12% SDS-PAGE and immunoblot analysis was carried out using anti--SM actin antibody. Lane 1, granulation tissue pouch; lane 2, cultured cells from the granulation tissue pouch; lane 3, cultured fibroblasts.

Full figure and legend (82K)

Induction of myofibroblast apoptosis by STS

To examine whether STS induces apoptosis in myofibroblasts, we performed flow cytometric analysis. Cells undergoing apoptosis expose phosphatidylserine on the outer surface membrane and are detected by annexin V binding (^{Walsh et al, 1998; Van Heerde et al, 2000}). Cells undergoing secondary necrosis, however, are not only positive in annexin V binding but also stain with PI. We counted cells stained with FITC-labeled annexin V as apoptotic cells. As shown in Figure 2a, b, apoptotic cells were significantly increased by STS treatment in a time- and a dose-dependent manner. Treatment with 10 nM of STS for 24 h induced apoptosis in approximately 50% of the cells, and the maximal effect was obtained at 1 M. Hoechst 33342 dye staining of the STS-treated cells showed nuclear shrinkage and chromatin condensation, morphologic changes typical of apoptotic cells (Figure 2c). These findings suggested that STS rapidly and strongly induced apoptosis in myofibroblasts.

Figure 2.

Myofibroblast apoptosis induced by STS. (A) Time course. Cells were treated with (closed circle) or without (open circle) 1 M STS for the indicated periods (0, 6, 12, 24 h) and stained with annexin V/FITC and PI for flow cytometry analysis. Values are means SEM of six independent experiments. ^**p<0.01 compared with at time 0 (Student' t test). (B) Dose dependence. Cells were treated with STS (0–1000 nM) for 24 h and stained with annexin V/FITC and PI for flow cytometry analysis. Values are means SEM of six independent experiments. ^**p<0.01 compared with at time 0 (Student' t test). (C) Nuclear staining with Hoechst 33342 dye. Cells were plated and grown on collagen-coated coverslips with or without 1 M STS for 3 h and stained with Hoechst 33342 dye. The results shown are representative of four independent experiments. Scale bar: 10 m.

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Activation of caspase-3 in STS-induced apoptosis

As caspase-3 is a key protease in the apoptosis signal transduction, whether STS activates caspase-3 was examined by Western blotting. Caspase-3 is usually present as a 32-kDa pro-form (pro-caspase-3). In response to proapoptotic signals, pro-caspase-3 is cleaved at 175^Asp followed by further cleavage at 9^Asp and 28^Asp, resulting in the production of the 20 kDa (p20), 19 kDa (p19), 17 kDa (p17), and 12 kDa (p12) active forms of this enzyme (^{Fernandes-Alnemri et al, 1996}). Treatment with STS decreased the amount of pro-caspase-3 and increased the active forms of caspase-3. The active forms of caspase-3 were increased at 3 h treatment with STS and reached maximal levels at 6 h (Figure 3a). To measure the activity of caspase-3, lysates from STS-treated myofibroblasts were assayed for caspase-3 activity using DEVD-p-nitroanilide as the substrate. The lysates from STS-treated myofibroblasts showed a greater than 3.6-fold increase in caspase-3 activity at 6 h treatment compared with the control and the activity declined to the baseline at 24 h (Figure 3b), consistent with the result shown in Figure 3(a). These results suggested that caspase-3 was activated by STS at an early stage of myofibroblast apoptosis.

Figure 3.

Effect of STS on caspase-3 in myofibroblasts. (A) Western blot analysis of casapase-3. Cells were treated with 1 M STS for the indicated periods (0, 1, 3, 6, 12, 24 h). Samples were separated by 15% SDS-PAGE and immunoblot analysis was carried out using anticaspase-3 antibody. Results are representative of three independent experiments. (B) Time course of caspase-3 activity induced by STS. Cells were treated with (closed circle) or without (open circle) 1 M STS for the indicated periods (0, 3, 6, 12, 24 h). Cell lysate was incubated with DEVD-p-nitroanilide at 37°C for 3 h and the samples were measured at 405 nm. Values are means SEM of three independent experiments. ^**p<0.01 compared with at time 0 (Student' t test).

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Degradation of -SM actin in STS-induced apoptosis

The progressive changes of -SM actin were observed in STS-treated myofibroblasts using immunofluorescence detection. The bundles of stress fibers composed of -SM actin were already indistinct after 1 h treatment with STS and were condensed in the periphery of the cells due to destruction after 6 h treatment (Figure 4a). We then carried out Western blotting to analyze the changes in the amount of -SM actin in myofibroblasts after STS treatment. As shown in Figure 4(b), (c), the amount of -SM actin was decreased as early as 1 h after the treatment with STS (64.0%18.9% of control; meanSEM, n=3). We also used an antibody specific to -actin and an antibody recognizing all actin isoforms (six known isoforms of actin including four muscle actins and two cytoplasmic actins) to investigate whether STS selectively degrades -SM actin. Both the level of -actin and the total amount of all the actin isoforms, however, were not significantly changed even after 6 h treatment (80.5%10.6% and 95.4%8.4% of control, respectively; meanSEM, n=3). These results suggested that -SM actin was selectively degraded in an early stage of the STS-induced apoptosis, in accordance with the time course of caspase-3 activation.

Figure 4.

-SM actin degradation induced by STS in myofibroblasts. (A) Time course of immunofluorescence staining with anti--SM actin antibody. Cells were plated and grown on collagen-coated coverslips. After 24 h incubation, cells were treated with 1 M STS for the indicated periods (0, 1, 6, 24 h) and fixed with 4% paraformaldehyde. Samples were probed with anti--SM actin antibody. The results are representative of three independent experiments. Scale bar: 20 m. (B) Western blot analysis with anti--SM actin antibody. Cells were treated with 1 M STS for the indicated periods (0, 1, 3, 6, 12, 24 h). Samples were separated by 12percnt; SDS-PAGE and immunoblot analysis was performed using anti--SM actin antibody. The results are representative of three independent experiments. (C) Quantification of -SM actin. The levels of -SM actin were quantified and shown as a percentage of the levels of -SM actin in control (time 0) cells. Values are means SEM of three independent experiments. ^**p<0.01 compared with at time 0 (Student' t test).

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Cleavage of purified -SM actin by caspase-3 in vitro

Actin is cleaved by activated caspase-3 in several types of cells (^{Chan and Mattson, 1999}). To clarify whether -SM actin in myofibroblasts is also cleaved by caspase-3, -SM actin purified from bovine aortic media was biotinylated and incubated with caspase-3 in vitro. We confirmed that the purified protein was -SM actin using anti--SM actin antibody (data not shown). As shown in Figure 5a, recombinant active caspase-3 cleaved -SM actin to 30 and 15 kDa fragments. We further investigated whether caspase-3 in STS-treated myofibroblasts also cleaves -SM actin in vitro. Caspase-3 was immunoprecipitated from myofibroblasts treated with 1 M of STS for 6 h and incubated with biotinylated -SM actin. Figure 5(b) shows that the immunoprecipitated caspase-3 cleaved -SM actin to 30 kDa fragments. These results suggested that -SM actin is the substrate for caspase-3 activated in the process of STS-induced apoptosis in myofibroblasts.

Figure 5.

Cleavage of purified -SM actin by activated caspase-3. (A) Recombinant human active caspase-3. Biotinylated -SM actin was incubated with or without recombinant active caspase-3 for 2 h at 37°C. Lane 1, biotinylated -SM actin alone; lane 2, biotinylated -SM actin + recombinant active caspase-3. (B) Immunoprecipitated caspase-3. Biotinylated -SM actin was incubated with immunoprecipitated caspase-3 from cells treated with or without STS for 6 h. Lane 1, biotinylated -SM actin + recombinant active caspase-3; lane 2, biotinylated -SM actin + immunoprecipitated caspase-3 from nontreated cells; lane 3, biotinylated -SM actin + immunoprecipitated caspase-3 from STS-treated cells. Samples were separated by 15% SDS-PAGE and the cleavage of -SM actin was detected with peroxidase-conjugated avidin. Values are representative of three independent experiments.

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Agents inducing myofibroblast apoptosis

Little is known about the physiologic stimuli leading to myofibroblast apoptosis. It has been reported that serum withdrawal, irradiation, and loss of adhesion to extracellular matrix could not induce apoptosis in cultured primary myofibroblasts (McKenna et al, 1996;^{McGill et al, 1997}). We investigated whether cytokines and growth factors, known to be involved in wound healing (^{Serini and Gabbiani, 1999}), induce myofibroblast apoptosis. Myofibroblasts treated with stimuli for 48 h in serum-free medium were analyzed by flow cytometry. As shown in Figure 6(a), anti-Fas antibody (100 ng per ml), TNF- (50 ng per ml), TGF-1 (100 ng per ml), and endothelin-1 (1 M) did not induce apoptosis even in the absence of serum. IFN- (1000 U per ml) and PDGF-BB (100 ng per ml) instead suppressed apoptosis. On the other hand, the synthetic chemical compounds bleomycin (100 ng per ml) and cisplatin (1 mM) induced apoptosis and activated caspase-3, although the effects of these compounds were much slower than STS (Figure 6a, b).

Figure 6.

Agents inducing myofibroblast apoptosis. (A) Flow cytometry analysis of myofibroblasts exposed to the stimulants. Cells were treated with STS (1 M), bleomycin (100 ng per ml), cisplatin (1 mM), anti-Fas monoclonal antibody (500 ng per ml), TNF- (50 ng per ml), TGF-1 (100 ng per ml), endothelin-1 (1 M), IFN- (1000 U per ml), or PDGF-BB (100 ng per ml) for 48 h in the absence of FBS. Values are means SEM of three independent experiments. **p<0.01 compared with control (without 10% FBS) (Student' t test). (B) Time course of the caspase-3 activity. Cells were treated with bleomycin (100 ng per ml), cisplatin (1 mM), and STS (1 M) for the indicated periods (0, 6, 12, 24, 48 h) in the absence of FBS. The cells were lysed on ice and the cell lysate was incubated with DEVD-p-nitroanilide at 37°C for 3 h. Samples were measured at 405 nm. Values are means SEM of three independent experiments: , control; , STS; , bleomycin; , cisplatin. ^*p<0.05, ^**p<0.01 compared with control (without 10% FBS) (Student' t test).

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Discussion

In this study, we showed that myofibroblasts undergo apoptosis by the treatment with STS, with typical morphologic changes such as nuclear shrinkage, chromatin condensation, and cell shrinkage. Mechanisms that cause these cell shape changes may involve the breakdown of cytoskeleton. In an attempt to address the relation between changes in cell morphology and those in cytoskeletal organization during apoptosis, we found that stress fibers mainly composed of -SM actin were disrupted, accompanied by the degradation of -SM actin at an early stage of STS-induced apoptosis. As -cytoplasmic actin is also highly expressed in myofibroblasts as well as -SM actin (^{Hinz et al, 2002}), we examined the effects of STS on the amount of -actin and the total amount of all actin isoforms. Their degradation was not significant at 6 h after the treatment with STS, however. Therefore, the proteolysis of -SM actin may selectively occur in an early stage of the STS-induced apoptosis in myofibroblasts.

Caspases have been reported to cleave actin in several types of cells (^{Chan and Mattson, 1999}). Actin is mainly cleaved at 244^Asp producing 30 and 15 kDa fragments (^{Kayalar et al, 1996; Mashima et al, 1997}). Amino acid sequences in actin isoforms are very well conserved and exhibit only small differences (less than 7%), which may relate to functional differences (^{Serini and Gabbiani, 1999}). As -SM actin also has a cleavage site for caspase-3 (at 246^Asp in humans and rats and at 244^Asp in bovine) (^{Vandekerckhove and Weber, 1979; McHugh and Lessard, 1988; Kamada and Kakunaga, 1989}), we tried to clarify whether caspase-3 is also able to degrade -SM actin. The anti--SM actin antibody used was expected to recognize 30 kDa fragments; however, we failed to detect the cleavage products in our experimental system. We do not know the precise reason for this failure, but the amount of cleaved endogenous -SM actin appeared to be too small to be detected by this antibody. Therefore, we employed biotinylated -SM actin for this analysis. As demonstrated, -SM actin, purified from bovine aorta and biotinylated, was cleaved not only by recombinant active caspase-3 but also by native caspase-3 immunoprecipitated from STS-treated myofibroblasts.

At this stage, however, the role of -SM actin cleavage in myofibroblast apoptosis is not clear. Further investigation is required to clarify this point. It is possible, however, that -SM actin cleavage during myofibroblast apoptosis may be required for the disappearance of granulation tissue, resulting in normal scar formation, and that disorder in this process might lead to the development of abnormal scar formation such as hypertrophic scar and keloid formation.

STS, originally isolated from Streptomyces as a potential antifungal agent, is known to be a potent inhibitor of protein kinase C. In this study, we showed that STS induced apoptosis through the activation of caspase-3 in myofibroblasts. Some studies have suggested that STS induces cellular apoptosis through the inhibition of protein kinase C (^{Jarvis et al, 1994; Ferraris et al, 1997}). Recently, however, it was reported that STS-induced apoptosis is protein kinase C independent (^{Han et al, 2000}). Whatever the involvement of protein kinase C, STS induces a release of cytochrome c from mitochondria to activate downstream caspase-3, resulting in apoptotic cell death (^{Desagher et al, 1999}).

Not only STS but bleomycin and cisplatin also induced myofibroblast apoptosis by activating caspase-3. Therefore, all of these compounds might have therapeutic value for abnormal scar formation. Indeed, intradermal bleomycin injection has been reported to be effective for the treatment of keloid and hypertrophic scars (^{Espana et al, 2001}). As STS induced apoptosis faster than the compounds we tested in this study, STS or its analogs might be more effective for these disorders than bleomycin and cisplatin. Indeed, the STS analog, N-benzoylated staurosporine, has been tested as an orally administrable anticancer agent in a phase I clinical trial (^{Propper et al, 2001}). It is possible that this agent is clinically applicable to abnormal scar formation such as hypertrophic scar and keloid formation.

References

1. Appleton, I, Tomlinson, A, Chander, CL, Willoughby, DA: Effect of endothelin-1 on croton oil-induced granulation tissue in the rata pharmacologic and immunohistochemical study Laboratory Invest 1992 67: 703–710,  | ISI | ChemPort |
2. Chan, SL, Mattson, MP: Caspase and calpain substratesRoles in synaptic plasticity and cell death J Neurosci Res 1999 58: 167–190,  | Article | PubMed | ISI | ChemPort |
3. Darby, I, Skalli, O, Gabbiani, G: -Smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Laboratory Invest 1990 63: 21–29,  | ISI | ChemPort |
4. Desagher, S, Osen-Sand, A, Nichols, A, et al: Bid-induced conformational change of Bax is responsible for mitchondrial cytochrome c release during apoptosis. J Cell Biol 1999 144: 891–901,  | Article | PubMed | ISI | ChemPort |
5. Desmouliere, A, Redard, M, Darby, I, Gabbiani, G: Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 1995 146: 56–66,  | PubMed | ISI | ChemPort |
6. Espana, A, Solano, T, Quintanilla, E: Bleomycin in the treatment of keloids and hypertrophic scars by multiple needle punctures. Dermatol Surg 2001 27: 23–27,  | Article | PubMed | ISI | ChemPort |
7. Fernandes-Alnemri, T, Armstrong, RC, Krebs, J, et al: In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc Natl Acad Sci USA 1996 93: 7464–7469,  | Article | PubMed | ChemPort |
8. Ferraris, C, Cooklis, M, Polakowska, RR, Haake, AR: Induction of apoptosis through the PKC pathway in cultured dermal papilla fibroblasts. Exp Cell Res 1997 234: 37–46,  | Article | PubMed | ISI | ChemPort |
9. Goto, T, Yanaga, F, Ohtsuki, I: Studies on the endothelin-1-induced contraction of rat granulation tissue pouch mediated by myofibroblasts. Biochim Biophys Acta 1998 1405: 55–66,  | PubMed | ISI | ChemPort |
10. Han, Z, Pantazis, P, Lange, TS, Wyche, JH, Hendrickson, EA: The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C. Cell Death Differ 2000 7: 521–530,  | Article | PubMed | ISI | ChemPort |
11. Hinz, B, Gabbiani, G, Chaponnier, C: The NH2-terminal peptide of -smooth muscle actin inhibits forece generation by the myofibroblast in vitro and in vivo. J Cell Biol 2002 157: 657–663,  | Article | PubMed | ISI | ChemPort |
12. Jacobson, MD, Burne, JF, Raff, MC: Mechanisms of programmed cell death and Bcl-2 protection. Biochem Soc Trans 1994 22: 600–602,  | PubMed | ISI | ChemPort |
13. Jarvis, WD, Turner, AJ, Povirk, LF, Traylor, RS, Grant, S: Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res 1994 54: 1707–1714,  | PubMed | ISI | ChemPort |
14. Kamada, S, Kakunaga, T: The nucleotide sequence of a human smooth muscle alpha-actin (aortic tipe) cDNA. Nucl Acids Res 1989 17: 1767,  | PubMed | ISI | ChemPort |
15. Kayalar, C, Ord, T, Testa, MP, Zhong, LT, Bredesen, DE: Cleavage of actin by interleukin 1-converting enzyme to reverse DNase I inhibition. Proc Natl Acad Sci USA 1996 93: 2234–2238,  | Article | PubMed | ChemPort |
16. Kerr, JFR, Wyllie, AH, Currie, AR: Apoptosisa basic biological phenomenon with wide-ranging implications in tissue kinetics Br J Cancer 1972 26: 239–257,  | PubMed | ISI | ChemPort |
17. Mashima, T, Naito, M, Noguchi, K, Miller, DK, Nicholson, DW, Tsuruo, T: Actin cleavage by CPP-32/apopain during the development of apoptosis. Oncogene 1997 14: 1007–1012,  | Article | PubMed | ISI | ChemPort |
18. Mashima, T, Naito, M, Tsuruo, T: Caspase-mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis. Oncogene 1999 18: 2423–2430,  | Article | PubMed | ISI | ChemPort |
19. McGill, G, Shimamura, A, Bates, RC, Savage, RE, Fisher, DE: Loss of matrix adhesion triggers rapid transformation-selective apoptosis in fibroblasts. J Cell Biol 1997 138: 901–911,  | Article | PubMed | ISI | ChemPort |
20. McHugh, KM, Lessard, JL: The nucleotide sequence of a rat vascular smooth muscle alpha-actin cDNA. Nucl Acids Res 1988 16: 4167,  | PubMed | ISI | ChemPort |
21. Paddenberg, R, Loos, S, Schoneberger, HJ, Wulf, S, Muller, A, Iwig, M, Mannherz, HG: Serum withdrawal induces a redistribution of intracellular gelsolin towards F-actin in NIH 3T3 fibroblasts preceding apoptotic cell death. Eur J Cell Biol 2001 80: 366–378,  | Article | PubMed | ISI | ChemPort |
22. Propper, DJ, McDonald, AC, Man, A, et al: Phase I and pharmacokinetic study of PKC412, an inhibitor of protein kinase C. J Clin Oncol 2001 19: 1485–1492,  | PubMed | ISI | ChemPort |
23. Raff, MC: Social controls on cell survival and cell death. Nature 1992 356: 397–400,  | Article | PubMed | ISI | ChemPort |
24. Serini, G, Gabbiani, G: Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res 1999 250: 273–283,  | Article | PubMed | ISI | ChemPort |
25. Spudich, JA, Watt, S: The regulation of rabbit skeletal muscle contraction. J Biol Chem 1971 246: 4866–4871,  | PubMed | ISI | ChemPort |
26. Vandekerckhove, J, Weber, K: The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscleA protein-chemical analysis of muscle actin differentiation Differentiation 1979 14: 123–133,  | PubMed | ISI | ChemPort |
27. Van Heerde, WL, Robert-Offerman, S, Dumont, E, et al: Markers of apoptosis in cardiovascular tissuesfocus on annexin V Cardiovasc Res 2000 45: 549–559,  | Article | PubMed | ISI | ChemPort |
28. Walsh, GM, Dewson, G, Wardlaw, AJ, Levi-Schaffer, F, Moqbel, R: A comparative study of different methods for the assessment of apoptosis and necrosis in human eosinophils. J Immunol Meth 1998 217: 153–163,  | Article | ISI | ChemPort |
29. Yamazaki, Y, Tsuruga, M, Zhou, D, et al: Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis. Exp Cell Res 2000 259: 64–78,  | Article | PubMed | ISI | ChemPort |

Acknowledgments

We thank Drs Hiroki Yoshida and Kozo Miyazaki, Medical Institute of Bioregulation, Kyushu University, for technical advice. This work was supported by the Uehara Memorial Foundation and a research grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.