Gelsolin, but not its cleavage, is required for TNF-induced ROS generation and apoptosis in MCF-7 cells

Biochemical and Biophysical Research Communications 385 (2009) 284–289

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Gelsolin, but not its cleavage, is required for TNF-induced ROS generation and apoptosis in MCF-7 cells Qinxi Li a,1,*, Zhiyun Ye a,1, Jun Wen a, Lin Ma b, Ying He a, Guili Lian a, Zhen Wang a, Luyao Wei a, Di Wu a, Bin Jiang a a b

The Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, 361005 Fujian, China Xiamen University Hospital, Xiamen, 361005 Fujian, China

a r t i c l e

i n f o

Article history: Received 10 May 2009 Available online 22 May 2009

Keywords: Gelsolin TNF-a Apoptosis ROS MCF-7

a b s t r a c t The tumor necrosis factor (TNF) can induce apoptosis in many cells including MCF-7 cells. To identify the genes responsible for TNF-induced apoptosis, we generated a series of TNF-resistant MCF-7 cell lines by employing retrovirus insertion-mediated random mutagenesis. In one of the resistant lines, gelsolin was found to be disrupted by viral insertion. Exogenous expression of gelsolin in this mutant cell line (Gelmut) restored the sensitivity to TNF-induced cell death and knock-down of gelsolin by siRNA conferred MCF-7 cells with resistance to TNF, indicating that gelsolin is required for TNF-induced apoptosis. Interestingly, the resistance of Gelmut cells to apoptosis induction is selective to TNF, since Gelmut and wild-type cells showed similar sensitivity to other death stimuli that were tested. Furthermore, TNF-induced ROS production in Gelmut cells was significantly decreased, demonstrating that gelsolin-mediated ROS generation plays a crucial role in TNF-induced apoptosis in MCF-7 cells. Importantly, caspase-mediated gelsolin cleavage is dispensable for TNF-triggered ROS production and subsequent apoptosis of MCF-7 cells. Our study thus provides genetic evidence linking gelsolin-mediated ROS production to TNF-induced cell death. Ó 2009 Elsevier Inc. All rights reserved.

Introduction TNF-a can induce apoptotic or necrotic cell death in vitro, depending on the nature of the cell line used [1,2]. MCF-7 human breast cancer cells undergo apoptosis in response to TNF-a treatment and are widely utilized to investigate the molecular mechanism underlying TNF-a-induced apoptosis [1,3]. Reactive oxygen species (ROS) generation in mitochondria is believed to play a key role in TNF-induced apoptosis [4,5]. However the intracellular molecules that link TNF stimulation to ROS production are not fully understood. Gelsolin is a protein which is 82–84 kDa, and it was initially identified in 1979 by Yin and Stossel [6]. Gelsolin contains six gelsolin-like (G) domains and controls actin organization by severing filaments, capping filaments ends and nucleating actin assembly [6–10]. In addition to its activity in the regulation of actin network, gelsolin has been reported to participate in the regulation of apoptotic process [11–13]. Gelsolin can be cleaved by caspase-3 at Asp 376, producing N-half and C-half fragments. The N-half fragment

can sever actin filaments independently of Ca2+ and dismantle the actin-based cytoskeleton to cause apoptosis [10]. However another study reported that caspase-3 is dispensable for the cleavage of gelsolin, indicating that other caspases may be able to cleave gelsolin [14]. Although increasing evidence has indicated that gelsolin is involved in apoptosis, the mechanism remains far from clear. We previously identified several genes that are essential for TNFa-induced cell death by using pDisrupt retrovirus insertion-mediated random mutagenesis [15–17]. Here, we have characterized another MCF-7-resistant cell line that carries retroviral insertion. In this cell line, gelsolin gene was disrupted by the retroviral vector insertion in exon 2. We thus termed this cell line as Gelmut. Gelmut cells showed strong resistance to TNF-induced apoptosis and exogenous expression of gelsolin restored the sensitivity of Gelmut cells to TNF stimulation, indicating that gelsolin is involved in TNF-induced apoptosis of MCF-7 cells. Further investigation suggested that gelsolin is required for TNF-a-induced ROS generation. Materials and methods

* Corresponding author. Address: The Department of Biomedical Sciences, School of Life Sciences, Xiamen University, 422, South Siming Road, Xiamen, 361005 Fujian, China. Fax: +86 592 218 4687. E-mail address: [email protected] (Q. Li). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.05.078

Reagents. Recombinant human TNF-a was purchased from Promega (Madison, WI). Propidium iodide (PI), H2O2, vincristine sulfate, mitomycin C and 5-fluorouracil were obtained from Sigma

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(St. Louis, MO). Hydroethidine (HE) and 20 ,70 -dichlorofluoresceindiacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, Oregan). Blasticidin S hydrochloride was obtained from Invitrogen (Carlsbad, CA). Goat gelsolin antibody (N-18) and mouse b-actin antibody (C4) were purchased from Santa Cruz Biotechnology. Cell culture and transfection. HeLa cells, U2OS cells, wild-type MCF-7 cells, Gelmut MCF-7 cells, and stable cell lines established from wild-type and Gelmut cells were all maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU penicillin, and 100 lg/ ml streptomycin at 37 °C in a humidified incubator containing 5% CO2. All transfections for the establishment of stable cell lines were performed using Lipofectamine 2000 transfection reagent. Retroviral mutagenesis screening. 30 rapid amplification of cDNA ends (RACE) technique was employed to identify the genes disrupted by virus insertion in TNF-resistant cell lines as described previously [17]. Briefly, total RNA was isolated using Trizol reagent (Invitrogen), followed by reverse transcription with RT primer (50 CCA GTG AGC AGA GTG ACG AGG ACT CGA GCT CAA GC[T]17-30 ). The cDNA was then amplified by nested PCR with primers: P1/Q1 (50 -AAA GCG ATA GTG AAG GAC AGT GA-30 and 50 -CCA GTG AGC AGA GTG ACG-30 ) and P2/Q2 (50 -TGC TGC CCT CTG GTT ATG TGT GG-30 and 50 -GAG GAC TCG AGC TCA AGC-30 ). P1 and P2 are located on the blasticidin+ gene, whereas Q1 and Q2 are located on the anchor sequence of RT. The endogenous gene that was fused with the blasticidin+ gene was determined by sequencing the PCR product. Measurement of cell survival rate. PI exclusion assay was used to determine the survival rates of TNF-treated cells. Briefly, cells were trypsinized, collected by centrifugation, washed once with phosphate-buffered saline (PBS), and resuspended in PBS containing 2 lg/ml PI. The levels of PI incorporation were quantified on a FACScan flow cytometer (EPICS XL; Beckman Coulter, Fullerton, CA). PI-negative cells with a normal size were considered as living cells. Measurement of ROS. ROS levels were determined by flow cytometry using the probe hydroethidine (HE) or dichlorofluorescein-diacetate (DCFH-DA). O2 can oxidize nonfluorescent HE to ethidium which gives a fluorescence emission that can be detected in the FL2 channel (575-nm filter). Nonfluorescent DCFH-DA is cleaved by nonspecific esterase to produce 20 ,70 -dichlorodihydrofluorescein (DCFH2) which in turn is oxidized by H2O2 to generate fluorescent 20 ,70 -dichlorofluorescein (DCF). The fluorescence emission of DCF can be detected in the FL1 channel (525-nm filter). TNF-treated or TNF-untreated cells were collected and washed once with PBS. The pellets were resuspended in PBS containing 2 lg/ml PI and 6.6 lM HE or 10 lM DCFH-DA, and were incubated for 30 min at 37 °C. PI-negative cells were gated and analyzed by FL2 or FL1 channel, respectively. RNA interference. The mammalian expression vector pSUPER was used for expression of gelsolin siRNA or control siRNA in MCF-7 cells. pSUPER-based interfering plasmid was constructed by inserting a 19-nucleotide sequence (50 -CAA TGG CGA CTG CTT CAT C-30 ) corresponding to human gelsolin sequence located 631 nucleotides downstream of the first ATG into the pSUPER vector. Control siRNA was expressed by the pSUPER vector carrying a 19-nucleotide scrambled sequence (50 -TCC TAC AAA TTC CAC AGT T-30 ). Western blotting. Cells were lysed in a lysis buffer (20 mM Tris–HCl, pH 7.5, 120 mM NaCl, 1 mM Na3VO4, 2 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, and 40 mM b-glycerophosphate) containing 1% Triton X-100. Cell lysates were separated on 10% SDS–polyacrylamide gels and were transferred to polyvinylidene difluoride membranes (Roche Diagnostics). After blocking with 5% skim milk in phosphate-buffered saline with 0.1% Tween 20 for 1 h, the membranes were probed with gelsolin antibody or actin antibody. All the data shown are representative results from at least three separate experiments.

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Results and discussion Gelsolin is required for TNF-a-induced apoptosis in MCF-7 cells To identify the genes required for TNF-a-induced apoptosis of MCF-7 cells, we established a retrovirus-mediated mutagenesis approach as described previously [17,18]. We first infected MCF-7 cells with pDisrupt retroviral vector carrying a blasticidin+ gene. This vector was designed such that the blasticidin+ gene is fused to the sequence of the exon located at the 30 end of the viral insertion site. Retroviral integration could generate mutant alleles leading to diminished or abolished expression of the target gene. TNF-resistant cell clones were obtained by sequential selection of infected cells with blasticidin and TNF-a, followed by determination of the disrupted genes using rapid amplification of cDNA ends (RACE) of the fused blasticidin+ mRNA. The gene disrupted in one of the TNF-resistant clones was identified as gelsolin and we termed this clone as Gelmut (Supplementary Fig. S1). Fig. 1A shows that the blasticidin+ gene encoded by the viral vector was inserted into the gelsolin coding region yielding a truncated protein that lacks the C-terminal region and therefore disrupted one allele of the gelsolin gene. Western blotting assays revealed a diminished expression of gelsolin protein (Fig. 1B), confirming that one allele of gelsolin gene had been disrupted. We then determined whether deficiency of gelsolin gene influences the sensitivity of Gelmut cells to TNF-induced apoptosis by using propidium iodide (PI) exclusion assay. Gelmut MCF-7 cell line exhibits obvious resistance to TNF-a-induced apoptosis in comparison with wild-type MCF-7 cells (Fig. 1C). To determine whether the resistance of Gelmut cells in response to TNF-a stimulation is due to reduced expression of gelsolin, we performed a reconstitution experiment by transfecting expression plasmid of gelsolin or empty vector into Gelmut cells and selected stable expression cells (designated as Gelmut + Gel and Gelmut + vector, respectively). As shown in Fig. 1D, a higher level of gelsolin was detected in Gelmut + Gel cells. These cells showed restored sensitivity to TNF-induced apoptosis; in contrast, Gelmut + vector cells showed the same resistance to TNF treatment as Gelmut cells, indicating that TNF resistance observed in Gelmut cells is due to a decreased expression of gelsolin (Fig. 1E). To confirm this finding, we generated cell lines stably expressing gelsolin siRNA by transfecting pSUPER-gelsolin (pSUPER-Gel) (C4, C5 and C6), along with cell lines (C1, C2 and C3) stably transfected with pSUPER carrying a scrambled 19-nucleotide sequence as control. Gelsolin protein levels were significantly reduced in cell lines C4, C5 and C6, compared with wild-type MCF-7 and control cell lines C1, C2 and C3 (Fig. 1F). Knock-down of gelsolin rendered the gelsolin siRNA-expressing MCF-7 cells resistant to TNF-induced apoptosis (Fig. 1G). These results demonstrate that gelsolin is indeed required for MCF-7 cell apoptosis triggered by TNF-a. We then investigated whether gelsolin plays the same role in other cell lines as in MCF-7. Surprisingly, knock-down of gelsolin endowed HeLa and U2OS cells with sensitivity to TNF-a-induced cell death (Fig. 1H and I), indicating that gelsolin plays an anti-apoptotic role in these two cell lines. Taken together, our results suggest that gelsolin can both positively and negatively regulate cell death depending on cell systems, which is consistent with previous reports showing that gelsolin plays an apoptotic role in neutrophils [10], and an anti-apoptotic role in HeLa and NIH3T3 cells [19].

The resistance of Gelmut cells is selective to TNF-a-induced cell death Because cell apoptosis triggered by different stimuli employs distinct or shared signaling transduction pathways, we then examined whether the resistance of Gelmut cells to apoptosis induction is

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Fig. 1. Gelsolin is required for TNF-a-induced apoptosis of MCF-7 cells. (A) The junction sequence of the cDNA derived from the mRNA fused between blasticidin+ and gelsolin is shown. The viral insertion occurred at the coding region of the gelsolin gene. The extreme C-terminal amino acid sequence of the product of blasticidin+ gene is shown beneath the corresponding cDNA sequence, in lowercase. The gelsolin sequence, in capitals, was inserted by retrovirus vector at nucleotide 501 (relative to the start codon of gelsolin), and is expected to be unable to be translated due to a stop codon (indicated by an asterisk) in viral sequence preceding the sequence. (B) Gelsolin expression was reduced in Gelmut cell line. Gelsolin from wild-type (Wt) MCF-7 cells and Gelmut cells was detected by Western blotting. b-Actin was probed as loading control. (C) Gelmut cells are resistant to TNF-induced cell death. Parental wild-type and Gelmut MCF-7 cells were treated with 50 ng/ml of TNF-a for different periods of time, followed by detection of survival rates by using flow cytometry based on propidium iodide (PI) staining. The results represented are the means ± SD (n = 3). (D) Western blotting was performed to determine the expression of transfected gelsolin in Gelmut cells, with b-actin detected as the loading control. (E) Reintroduction of gelsolin confers Gelmut cells with the sensitivity to TNF-induced cell death. The viabilities of Gelmut, Gelmut + vector and Gelmut + Gel cells in response to TNF stimulation were measured as in (C). (F) Gelsolin protein levels were detected by Western blotting in wild-type MCF-7 cells, stable cell lines (C1, C2 and C3) expressing control siRNA and cell lines stably expressing gelsolin siRNA (C4, C5 and C6). Expression levels of b-actin in these cell lines were measured as loading control. (G) Knock-down of gelsolin renders MCF-7 cells resistant to TNFinduced cell death. The survival rates of these stable cell lines were determined as in (C). Gelsolin plays an anti-apoptotic role in HeLa (H) and U2OS (I) cells upon TNF stimulation. HeLa and U2OS cells that stably express pSUPER-Gel or control siRNA were treated with different doses of TNF-a for 36 h and survival rates were assessed as in (C).

selective to TNF-a. As shown in Fig. 2A, Gelmut cells were resistant to cell death induced by various doses of TNF treatment, which was consistent with our observation in Fig. 1C. However, Gelmut and wild-type MCF-7 cells showed similar sensitivity to UV (ultraviolet) stimulation (Fig. 2B). The sensitivity of Gelmut cells to H2O2 (Fig. 2C)-, vincristine (Fig. 2D)-, mitomycin C (Fig. 2E)- and 5-fluorouracil (Fig. 2F)-induced cell death was also comparable with that observed in the wild-type MCF-7 cells. Therefore, TNF resistance caused by gelsolin deficiency is not due to the disruption of a general pathway used by death stimuli, but is caused by the impairment of the death pathway specifically used by TNF. Gelsolin is essential for TNF-induced ROS generation in MCF-7 cells It has been shown that ROS generation plays a crucial role in TNF-induced apoptosis [4,5]. We asked whether gelsolin deficiency affects TNF-induced production of ROS. HE is nonfluorescent and can be oxidized to the fluorescent ethidium by O2, and thus it is widely used to detect intracellular O2 level by determining the fluorescence intensity of ethidium. DCFH-DA is

a cell-permeable and nonfluorescent compound. It can be cleaved by cellular esterases to DCFH2 that is further oxidized to fluorescent DCF by H2O2 only in the presence of a catalyst such as peroxidase, cytochrome, and Fe3+. Therefore this reagent is frequently used to determine the relative H2O2 levels and peroxidase activity of target cells by reading the fluorescent intensity of its oxidized product DCF. We employed HE and DCFH-DA to measure the redox state of MCF-7 and Gelmut cells. As shown in Fig. 3A, a representative result of three independent experiments, increased production of O2 (Fig. 3A, left panel) and H2O2 (Fig. 3A, right panel) was detected in TNF-treated wild-type cells (red lines) in a time-dependent manner. In contrast, the induction of ROS was dramatically diminished in Gelmut cells (blue lines), indicating that gelsolin is required for TNF-induced ROS generation in MCF-7 cells. The corresponding statistical results of Fig. 3A are shown in Supplementary Fig. S2. To confirm this finding, we examined the ROS production in Gelmut + Gel cells. Reintroduction of gelsolin expression in Gelmut cells restored the sensitivity to TNF-induced O2 (Fig. 3B, left panel) and H2O2 (Fig. 3B, right panel) production. Consistently, knock-down of gelsolin by pSUPER-

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Fig. 2. Gelmut cells are selectively resistant to TNF-a-induced cell death. (A) Parental wild-type and Gelmut cells were treated with different doses of TNF-a for 36 h and survival rates were assessed by PI exclusion assay. Results are presented as the means ± SD (n = 3). (B) Wild-type and Gelmut cells were exposed to different doses of UV irradiation, and at 8 h after treatment survival rates were measured as in (A). Wild-type and Gelmut cells were treated with various concentrations of H2O2 (C), vincristine (D), mitomycin C (E) and 5-fluorouracil (F) for 18, 96, 96, and 96 h, respectively. Cell survival rates were determined as in (A).

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Fig. 3. Gelsolin is involved in TNF-induced ROS generation. (A) Wild-type and Gelmut cells were treated with TNF (50 ng/ml) for different periods of time as indicated, followed by staining with HE and DCFH-DA for measurement of O2 and H2O2 levels, respectively. Fluorescent intensities of ethidium, the oxidized product of HE, and DCF, the fluorescent product of DCFH-DA were determined by flow cytometry. A representative result of three independent experiments is shown. The corresponding statistical results are presented in Supplementary Fig. S2. (B) Exogenous expression of gelsolin restores the sensitivity of Gelmut cells to TNF-induced ROS generation. TNF-treated Gelmut, Gelmut + vector, and Gelmut + Gel cells were stained with HE and DCFH-DA. ROS production was determined as in (A). Results are presented as the means ± SD (n = 3). (C) Knock-down of gelsolin endows the Wt MCF-7 cell with resistance to TNF-induced ROS generation. Stable cell lines separately expressing pSUPER-based gelsolin siRNA and control siRNA were treated with 50 ng/ml of TNF for different periods of time as indicated. ROS production was detected as in (A).

based siRNA significantly decreased the generation of O2 (Fig. 3C, left panel) and H2O2 (Fig. 3C, right panel) in MCF-7 cells. Collectively, our data demonstrate that gelsolin is involved in TNF-induced ROS production. Since ROS induction is essential for TNF-stimulated apoptosis of MCF-7 cells, the resistance to TNF caused by gelsolin disruption most likely results from the blocking of TNF-induced ROS generation. Importantly, gelsolin deficiency in Gelmut cells can block apoptosis induced by exogenous H2O2 (Fig. 2C), indicating that gelsolin acts upstream of ROS generation in the TNF-induced cell death pathway.

Gelsolin cleavage is dispensable for TNF-induced MCF-7 apoptosis It has been shown that gelsolin can be cleaved by caspase-3 to produce 39- and 41-kDa products, and this cleavage makes an important contribution to the apoptotic morphological change of neutrophils upon TNF treatment [10]. However, another study indicated that caspase-3 is dispensable for cleavage of gelsolin [14], as in MCF-7 cells that are defective for caspase-3 expression owning to the functional deletion of the CASP-3 gene [20], gelsolin was still cleaved during apoptosis triggered by Fas antibody or TNF.

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Fig. 4. Gelsolin cleavage is not required for ROS production or apoptosis of MCF-7 cells in response to TNF stimulation. (A) Wild-type and Gelmut cells were treated with TNF (50 ng/ml) for different periods of time as indicated. Gelsolin and b-actin were detected by Western blotting. No cleaved gelsolin N-half fragment (41 kDa) was detected by using gelsolin antibody (N-18) that can recognize the full-length and N-half of gelsolin but not the C-half of gelsolin). (B) Generation of Gel-DM in which the two aspartic acid residues in the caspase-3 cleavage site on gelsolin were mutated into glycine. (C) Gelmut-based cell lines stably transfected with Gel-DM or vector were detected for gelsolin expression. (D) The sensitivity to TNF-induced apoptosis was restored by Gel-DM in Gelmut cells. Gelmut, Gelmut + vector and Gelmut + Gel-DM cells were treated with 50 ng/ml of TNF-a for different periods of time as indicated, followed by measurement of cell survival rates. Results that are shown are the means ± SD of three independent experiments. (E) Gelmut, Gelmut + vector and Gelmut + Gel-DM cells were treated with TNF (50 ng/ml) for a time course, followed by staining with HE or DCFH-DA. The fluorescent intensities of ethidium and DCF were measured to show the levels of O2 and H2O2, respectively. Results that are shown are means ± SD (n = 3).

This controversy promoted us to investigate whether gelsolin is indeed cleaved by other caspases in caspase-3-null MCF-7 cells in response to TNF stimulation, and if so whether the cleavage is required for TNF-induced gelsolin-mediated MCF-7 cell apoptosis. As shown in Fig. 4A, we never detected the gelsolin protein corresponding to the N-half fragment by using the gelsolin antibody (N18) that can detect full-length and N-half of gelsolin in MCF-7 cells treated with 50 ng/ml of TNF for the time course indicated, demonstrating that gelsolin cannot be cleaved by other caspases in caspase-3-depleted MCF-7 cells, which is consistent with the report showing that caspase-3 is the unique executioner of gelsolin [10,21]. Because caspase-3 and caspase-7 are similar in their specificities in recognizing primary cleavage sites, some substrates cleaved by caspase-3 will also be cleaved by caspase-7. To exclude the possibility that the trace amount of gelsolin cleaved by caspase-7 is below the detection limit of our Western blotting assays, we generated a gelsolin mutant Gel-DM that is defective for caspase-3 cleavage, in which two aspartic acid residues situated in the caspase cleavage site were mutated to glycine (Fig. 4B). We transfected Gel-DM or empty vector into Gelmut cells and established Gelmut + Gel-DM cell line stably expressing Gel-DM and control cell line Gelmut + vector (Fig. 4C). Gelmut + Gel-DM cells reconstituted the sensitivity to TNF-induced cell death, demonstrating that gelsolin cleavage is not essential for MCF-7 cell apoptosis triggered by TNF (Fig. 4D). Consistently, expression of Gel-DM can restore the ability of Gelmut cells to generate ROS upon TNF treatment (Fig. 4E). In summary, our data have provided a strong evidence that gelsolin is required for TNF-induced apoptosis, and that its cleavage is dispensable for TNF-induced ROS generation and subsequent apoptosis of MCF-7 cells.

Acknowledgments We thank Dr. Jiahuai Han for providing MCF-7 C2 cell line and Dr. Sheng-Cai Lin for critical reading. This work was supported by grants from the ‘‘973 Program” (#2007CB914602, #2006CB503900), and by grants from National Natural Science Foundation of China (#30770454 and #30730025) and NCETXMU. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.05.078. References [1] W. Fiers, R. Beyaert, W. Declercq, P. Vandenabeele, More than one way to die: apoptosis, necrosis and reactive oxygen damage, Oncogene 18 (1999) 7719– 7730. [2] M. Tewari, V.M. Dixit, Recent advances in tumor necrosis factor and CD40 signaling, Curr. Opin. Genet. Dev. 6 (1996) 39–44. [3] A. Strasser, L. O’Connor, V.M. Dixit, Apoptosis signaling, Annu. Rev. Biochem. 69 (2000) 217–245. [4] V. Goossens, J. Grooten, K. De Vos, W. Fiers, Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity, Proc. Natl. Acad. Sci. USA 92 (1995) 8115–8119. [5] K. Schulze-Osthoff, A.C. Bakker, B. Vanhaesebroeck, R. Beyaert, W.A. Jacob, W. Fiers, Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation, J. Biol. Chem. 267 (1992) 5317–5323. [6] H.L. Yin, T.P. Stossel, Control of cytoplasmic actin gel–sol transformation by gelsolin, a calcium-dependent regulatory protein, Nature 281 (1979) 583–586. [7] H.L. Yin, Gelsolin: calcium- and polyphosphoinositide-regulated actinmodulating protein, Bioessays 7 (1987) 176–179. [8] H.Q. Sun, M. Yamamoto, M. Mejillano, H.L. Yin, Gelsolin, a multifunctional actin regulatory protein, J. Biol. Chem. 274 (1999) 33179–33182.

Q. Li et al. / Biochemical and Biophysical Research Communications 385 (2009) 284–289 [9] P. Silacci, L. Mazzolai, C. Gauci, N. Stergiopulos, H.L. Yin, D. Hayoz, Gelsolin superfamily proteins: key regulators of cellular functions, Cell. Mol. Life Sci. 61 (2004) 2614–2623. [10] S. Kothakota, T. Azuma, C. Reinhard, A. Klippel, J. Tang, K. Chu, Caspase-3generated fragment of gelsolin: effector of morphological change in apoptosis, Science 278 (1997) 294–298. [11] L. Mullauer, H. Fujita, A. Ishizaki, N. Kuzumaki, Tumor-suppressive function of mutated gelsolin in ras-transformed cells, Oncogene 8 (1993) 2531–2536. [12] H. Tanaka, R. Shirkoohi, K. Nakagawa, H. Qiao, H. Fujita, F. Okada, J. Hamada, S. Kuzumaki, M. Takimoto, N. Kuzumaki, siRNA gelsolin knockdown induces epithelial–mesenchymal transition with a cadherin switch in human mammary epithelial cells, Int. J. Cancer 118 (2006) 1680–1691. [13] S. Moriya, K. Yanagihara, H. Fujita, N. Kuzumaki, Differential expression of HSP90, gelsolin and GST-p in human gastric carcinoma cell lines, Int. J. Oncol. 5 (1994) 1347–1351. [14] R.U. Ja nicke, P. Ng, M.L. Sprengart, A.G. Porter, Caspase-3 is required for aFodrin cleavage but dispensable for cleavage of other death substrates in apoptosis, J. Biol. Chem. 273 (1998) 15540–15545. [15] J. Li, Q. Li, C. Xie, H. Zhou, Y. Wang, N. Zhang, H. Shao, S.C. Chan, X. Peng, S.C. Lin, J. Han, b-Actin is required for mitochondria clustering and ROS generation

[16]

[17]

[18] [19]

[20]

[21]

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in TNF-induced, caspase-independent cell death, J. Cell Sci. 117 (2004) 4673– 4680. C. Xie, N. Zhang, H. Zhou, J. Li, Q. Li, T. Zarubin, S.-C. Lin, J. Han, Distinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells, Mol. Cell. Biol. 25 (2005) 6673–6681. C.J. da Silva, Y. Miranda, N. Austin-Brown, J. Hsu, J. Mathison, R. Xiang, H. Zhou, Q. Li, J. Han, R.J. Ulevitch, Nod1-dependent control of tumor growth, Proc. Natl. Acad. Sci. USA 103 (2006) 1840–1845. X. Wang, K. Ono, S.O. Kim, V. Kravchenko, S.C. Lin, J. Han, Metaxin is required for tumor necrosis factor-induced cell death, EMBO Rep. 2 (2001) 628–633. H. Kusano, S. Shimizu, R.C. Koya, H. Fujita, S. Kamada, N. Kuzumaki, Y. Tsujimoto, Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC, Oncogene 19 (2000) 4807–4814. F. Li, A. Srinivasan, Y. Wang, R.C. Armstrong, K.J. Tomaselli, L.C. Fritz, Cellspecific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release, J. Biol. Chem. 272 (1997) 30299– 30305. E.A. Slee, C. Adrain, S.J. Martin, Executioner caspase-3, -6 and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis, J. Biol. Chem. 276 (2001) 7320–7326.