ToxSci Advance Access originally published online on November 20, 2006
Toxicological Sciences 2007 96(1):83-91; doi:10.1093/toxsci/kfl172
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Cellular Toxicity Induced by SRF-Mediated Transcriptional Squelching
Department of Biochemistry, SUNY at Buffalo, Buffalo, New York 14214
1 To whom correspondence should be addressed at Department of Biochemistry, SUNY at Buffalo, 3435 Main Street, Buffalo, NY 14214. Fax: (716) 829-3106. E-mail: chunglee{at}buffalo.edu.
Received September 25, 2006; accepted November 14, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The transcriptional activator serum response factor (SRF) is a member of the immediate early gene family known to promote embryonic development, cell growth, and myogenesis through interaction with multiple nuclear protein factors. Previous studies have shown that SRF possesses potent transcriptional activation domains that can interfere with gene expression at artificially high expression levels through "transcriptional squelching." The current work sought to characterize toxicological aspects of SRF-mediated transcriptional squelching. An adenoviral expression system driven by the potent cytomegalovirus promoter was used to achieve up to a 50-fold increase in SRF protein levels. The overexpressed SRF is nuclear localized and interferes with gene expression independent of specific promoter interaction as expected for transcriptional squelching. SRF-mediated squelching elicits robust cell killing affecting multiple cell types including normal and abnormal proliferating cells as well as postmitotic cells such as cardiomyocytes in culture, and the cell killing is more pronounced than that mediated by the tumor suppressor protein p53. Although both the DNA-binding and transcriptional activation domains of SRF are normally required for the physiological roles of SRF, only the transcriptional activation domain is required for cell killing. Unlike c-myc–induced cell killing, squelching-induced cell death does not require serum withdrawal and cannot be effectively attenuated by blocking the caspase and calpain proteolytic pathways or by overexpression of the antiapoptotic gene bcl-xL. These findings suggest transcriptional squelching may be engineered for killing cancer cells, and the SRF gene may represent a novel molecular target for cancer therapeutics.
Key Words: SRF; squelching; cellular toxicity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Over the past decade, adenovirus-based gene expression vectors have been used extensively for gene toxicity studies, and genes explored toward tumor cell killing include tumor-suppressing genes, proapoptotic and suicide genes, and antiangiogenic genes (Cory and Adams, 2005
; Kaplan, 2005). These genes exert their cytotoxic effects in part by modulating the expression of growth, differentiation, and survival control genes. The tumor suppressor gene p53, for example, is well known for its cell death–inducing property upon overexpression, and this cytotoxicity of p53 is mediated through fine-tuning the bcl-2 rheostat in some cell systems (Leri et al., 1997; Miyashita et al., 1994). In addition, cytotoxicity and genotoxicity of many compounds or drugs are known to be directed against components of the transcription machinery (Bachman et al., 2006; Peraza et al., 2006). Thus, the cellular transcription machinery may represent an ideal target for therapeutic tumor cell killing.
The transcription machinery functions through an intricate network of general and sequence-specific transcription factors forming dynamic highly regulated multiprotein complexes within the genome. Transcriptional activation in particular hinges on multiple protein interactions directed by DNA sequence–specific transcriptional activators and involving coactivators, basal transcription factors, and RNA polymerase (Ranish and Hahn, 1996). It has been observed that experimental artifacts can arise from artificially introduced high levels of a potent transcriptional activator causing nonspecific transcriptional suppression, and this phenomenon is often referred to as transcriptional squelching (Gill and Ptashne, 1988; Natesan et al., 1997). Transcriptional squelching is thought to result from titration of one or more essential transcription factors present in limiting amounts by the abundance of the overexpressed transcriptional protein activation domain. Although transcriptional squelching can apparently interfere with gene expression and potentially affect normal cell function, little information exists regarding its cellular toxicity.
Serum response factor (SRF) is a potent transcriptional activator known to regulate numerous genes associated with cell growth and differentiation (Chai and Tarnawski, 2002; Lee et al., 1991, 1992; Miano, 2003). These diverse functions of SRF are mediated in part by the recruitment and activation of various members of the SRF coactivator family such as p62TCF and tissue-specific myocardin/megakaryoblastic leukemia (Cen et al., 2003; Gille et al., 1992; Wang et al., 2002). In addition, SRF is known to interact with many general and sequence-specific transcription factors, highlighting the central role of protein interaction in transcriptional regulation by SRF (Moore et al., 2001; Ramirez et al., 1997; Sartorelli et al., 1990). Indeed, high-level expression of SRF has been shown by us and others to cause abnormal gene suppression through transcriptional squelching (Lee et al., 1992, 1998; Prywes and Zhu, 1992). The work presented here reveals for the first time that SRF-mediated transcriptional squelching exerts severe cytotoxicity, and the extent of cell killing is more pronounced than that mediated by high-level expression of the tumor suppressor gene p53. Consistent with this demonstration, squelching-mediated cytotoxicity is dependent on the transcription activation (or protein interaction) domain but not the DNA-binding domain of SRF. Additional experiments are presented showing that cytotoxicity caused by transcriptional squelching is largely independent of the caspase and calpain proteolytic pathways. Our study suggests that SRF-mediated transcriptional squelching may be engineered as a module for cell killing.
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MATERIALS AND METHODS |
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Cell culture.
Porcine bone marrow–derived mesenchymal stem cells (MSCs) were isolated as described (Vacanti et al., 2005
). MSCs were maintained in advanced Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS) and 50 µg/ml gentamycin and incubated in a humidified 5% CO2 atmosphere at 37°C. The MSC cultures used in the study received less than 15 passages. Neonatal rat cardiomyocytes were prepared as previously described (Kositprapa et al., 2000), and the cells were maintained in MEM supplemented with 10% horse serum human embryonic kidney (HEK) cells, mouse Sol8 myoblasts, P19 embryonal carcinoma cells, and HIIEC3 hepatoma cells were maintained in MEM supplemented with 5% FBS. Cells were trypsinized using a 0.05% trypsin-0.5mM ethylenediaminetetraacetic acid solution upon confluency.
Recombinant adenovirus.
The recombinant adenovirus expressing the human SRF cDNA (Ad-SRF) was constructed using the pAdEasy-1 system (He et al., 1998). A HindIII-NotI DNA fragment containing the full-length human SRF cDNA sequence was inserted into the pShuttle-cytomegalovirus (CMV) vector. The recombinant shuttle DNA and pAdEasy-1 DNA were recombined in HEK293 cells. The virus was amplified three rounds in HEK293 cells to yield 
109 viral particles per milliliter culture medium. Three SRF deletion mutants (Lee et al., 1992) were also expressed using the adenovirus system. The SRF DM1 mutant cDNA encoded by an NdeI (Klenow filled)-XbaI DNA fragment was inserted into pShuttle-CMV. The SRF DM3 and DM5 mutant cDNAs each encoded by a KpnI-XbaI DNA fragment were inserted into pShuttle-CMV. Recombinant virus was confirmed by reverse transcriptase–polymerase chain reaction and Western blotting analysis. Ad-p62TCF was constructed by inserting a KpnI-XbaI p62TCF cDNA into pShuttle-CMV. Ad-YAF2 was constructed by inserting a HindIII-EcoRV YAF2 cDNA into pShuttle-CMV. Ad-Hath1/green fluorescence protein (GFP) was provided by Richard Salvi (University at Buffalo). Ad-LacZ was provided by Kirk Hammond (University of California, San Diego). The reporter virus encodes a nuclear-localized ß-galactosidase (Giordano et al., 1996). Ad-p53 was a gift from Bert Vogelstein (Johns Hopkins University). Ad-IC encodes rat calpastatin, an inhibitor of calpain (IC), and its construction was documented in our previous work (Lin et al., 2004). Ad-Bcl-xL, which encodes the porcine antiapoptotic protein Bcl-xL, was constructed by inserting a SalI-NotI bcl-xL cDNA fragment into the shuttle vector. For cell infection, viral lysates were diluted 10-fold with serum-free MEM and added to adherent cells for 2–3 h with occasional agitation, following which cells were washed and maintained in the growth medium.
Plasmid construction.
The pVEGF-Luc vector was provided by Amit Maity (University of Pennsylvania), in which expression of the luciferase gene is driven by an 1.5-kb vascular endothelial growth factor (VEGF) promoter fragment from – 1174 to + 338 (Maity et al., 2000). The c-fos promoter–luciferase reporter construct (pFos-Luc) contains a 400-bp c-fos promoter fragment isolated from pFos-Cat using SalI (Klenow filled) and HindIII; the promoter fragment was inserted into pGL2-Basic (Promega, Madison, WI) digested with SmaI and HindIII. The serum response element (pSRE)-Luc contains a synthetic SRE oligonucleotide flanked by KpnI and MluI, which was inserted into the corresponding sites upstream from the thymidine kinase promoter TATA box derived from the vector pBLCAT2 (Luckow and Schutz, 1987). The top and bottom strands of the SRE oligonucleotides are CCCCTTACACAGGATGTCCATATTAGGACATCTGCGTCAGCAGGA and CGCGTCCTGCTGACGCAGATGTCCTAATATGGACATCCTGTGTAAGGGGGTAC, respectively.
Transient DNA transfection and luciferase assays.
Sol8 myoblasts were plated onto 35-mm dishes on the day before transfection. Cells were first exposed to the adenoviral lysate for 1 h, following which preformed calcium phosphate–DNA crystals were added to the dishes. Cells were then incubated with both the virus and DNA crystals for an additional 3 h, following which cells were washed twice and refed with a regular growth medium. Calcium phosphate–DNA crystals were prepared as described previously (Lee et al., 1998). The crystals were allowed to form at room temperature for 1 h prior to being added to the cells. Luciferase assays were performed 20 h after transfection as described (Lee et al., 1998). Typically 20 µl cell lysate was mixed with 100 µl luciferin solution, and relative light units (RLUs) were recorded for 10 s after mixing by Lumat LB9501. RLUs presented were normalized with total lysate protein concentrations.
MTT cell viability and LDH release assays.
Cells were plated onto 24-well plates (2 x 104 cells/well) in regular growth media for the assays. Protocols for MTT and LDH release assays were as described previously (Chen et al., 1998). In brief, growth medium containing 0.25 mg/ml MTT was added to each well, and cells were further incubated at 37°C for 20 min, following which the medium was replaced by 0.2 ml dimethyl sulfoxide (DMSO) per well. MTT reduction was determined by measuring the optical density (O.D.)540 nm of the DMSO extracts using DMSO as blank. Cell death as determined by lactic dehydrogenase (LDH) release was performed by mixing 20 µl collected culture medium with 50 µl LDH assay solution in a 96-well plate. The plate was incubated at 37°C for 30–60 min, following which the assay was terminated by addition of 50 µl 5% acetic acid to each well. LDH activities in the media were measured at O.D.492 nm. Some error bars were too small to appear on the graph.
In situ staining.
Cells were plated onto glass cover slips placed in 35-mm dishes. Cells were infected after overnight plating and fixed 1–2 days after viral infection by 4% paraformaldehyde. Fixed cells were immersed in a phosphate-buffered saline (PBS) solution supplemented with 0.2% Triton X-100, 2% horse serum, and 1% bovine serum albumin (BSA) for 1 h at room temperature. Cells were then incubated with SRF antibody (MacLellan et al., 1994) diluted 200-fold in PBS supplemented with 1% BSA at room temperature for 3 h. After washing, cells were probed with an FITC-conjugated secondary antibody for 1 h. X-gal staining of ß-galactosidase was performed by immersing fixed cells in the substrate solution (60mM Hepes, pH 7.4, 100mM NaCl, 3mM MgCl2, 3mM potassium ferricyanide, 3mM potassium ferrocyanide, and 0.5 mg/ml X-gal) at 37°C for several hours until the blue color appeared. Digital imaging was performed using a Nikon E600 fluorescence microscope.
Western blotting.
Total cellular proteins (typically 20–30 µg per lane) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to Immobilon-P membrane as described (Walowitz et al., 1998). The membrane was first incubated with a 1000-fold diluted primary antibody solution for 3 h followed by washing with a PBS solution supplemented with 0.025% TW-20. Secondary antibodies conjugated with horse radish peroxidase were used to probe blotted membranes, and signals were developed using the chemiluminescent substrate luminol (Pierce Biotechnology, Rockford, IL) and imaged by Fuji imager. The LacZ antibody (clone GAL-40) was purchased from Sigma, St Louis, MO.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenoviral Expression of SRF
Adenovirus-based vectors have been used extensively for gene transfer and cancer gene therapy (Cory and Adams, 2005
; Kaplan, 2005). We used the replication-deficient pAdEasy-1 adenovirus system (He et al., 1998) to study toxicological aspects of SRF-mediated transcriptional squelching. Expression of the human SRF cDNA is driven by the constitutive cytomegalovirus promoter. Since SRF is a nuclear-localized protein (Misra et al., 1991), we also used a nuclear-localized ß-galactosidase as control (Ad-LacZ) for the study. Western blotting using a monoclonal ß-galactosidase antibody shows increasing levels of ß-galactosidase in Ad-LacZ–infected MSCs only (Fig. 1, top left panel). We also performed activity staining using the chromogenic ß-galactosidase substrate X-gal, which reveals the nuclear localization of the reporter enzyme (Fig. 1, top right panel). Immunostaining of uninfected cells using an SRF antibody revealed the endogenous SRF protein primarily localized in the nuclei, and some weak cytoplasmic SRF staining was evident with a long exposure time (Fig. 1, bottom left panel). Cells infected with Ad-SRF exhibited intensely strong nuclear-localized SRF signals readily captured with a short exposure time (Fig. 1, bottom right panel). High-level SRF expression was also confirmed by Western blotting (see below).
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Transcriptional Squelching by Adenovirus-Expressed SRF
We next determined whether the recombinant adenovirus–introduced high levels of SRF might interfere with promoter function through transcriptional squelching as observed in previous in vitro and transient transfection assays (Lee et al., 1992
, 1998; Prywes and Zhu, 1992). We performed transient transfection analysis of a panel of promoter-reporter constructs. The immediate early gene c-fos, which carries a single promoter SRF-binding site termed SRE, is normally activated by SRF (Shaw et al., 1989). A pFos-Luc was examined here along with an artificial luciferase reporter construct driven by the single c-fos SRE (pSRE-Luc). In addition, a VEGF promoter construct, which does not contain any known SRE, was analyzed in parallel. Cells were transfected with the luciferase reporter DNA immediately following adenovirus infection. Luciferase assays show that adenoviral expression of SRF nonspecifically repressed the activities of both SRE-containing and SRE-lacking promoter targets, as expected for transcriptional squelching (Fig. 2). The inhibitory effect on the VEGF promoter is consistent with the notion that transcriptional squelching can suppress a promoter target independent of specific DNA-protein interaction.
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Cellular Toxicity Caused by Transcriptional Squelching
We then examined whether SRF-mediated transcriptional squelching might lead to substantial cell death using the MTT cell viability assay (Vacanti et al., 2005
). Figure 3 (left panel) shows that no difference in cell growth kinetic between LacZ- and SRF-expressing cells was observed during the first 24 h after infection. Cell loss in the SRF expressors first became evident at 48 h and much more severe at 72 h, consistent with increasing SRF protein levels 1–3 days after infection (data not shown). This analysis documents a hitherto unrecognized cytotoxic effect of SRF-mediated transcriptional squelching. SRF appeared quite potent in triggering cell killing since adenoviral expression of three other transcription factors did not lead to cell death (Fig. 3, right panel). MTT assays show that adenoviral expression of YAF2, a promyogenic transcription factor (Kalenik et al., 1997), Hath1, a neurogenic transcription factor (Lee, 1997), and p62TCF, an SRF coactivator (Gille et al., 1992), exhibited no inhibitory effect on cell viability. Interestingly, exogenous p62TCF alone modestly promoted the growth of MSCs during the 4-day period examined. In spite of its growth-promoting effect, expression of p62TCF was unable to attenuate the cytotoxicity caused by high levels of SRF.
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Cell Killing Requires the Transcription Activation Domain of SRF
Transcriptional squelching is mediated by protein-protein interaction and is not thought to require direct DNA-protein interaction (Gill and Ptashne, 1988
; Natesan et al., 1997). If the observed cell killing is indeed triggered by SRF-mediated transcriptional squelching, it can be expected that an SRF mutant lacking its transcription activation domain would exhibit reduced cytotoxicity, and a mutant lacking its DNA-binding domain would still manifest cytotoxicity. Three SRF deletion mutants (DM1, DM3, and DM5) constructed by us previously (Lee et al., 1992) were further expressed by the adenoviral vector. DM1 lacks amino acids 54–114 removing the alanine- and glutamate-rich domains and several protein phosphorylation sites (Fig. 4, top panel). DM3 lacks amino acids 10–72 and amino acids 141–172, thus lacking an intact DNA-binding domain. DM5 contains the N-terminal half of the protein, lacking the C-terminal amino acids 246–508, which is required for SRF-mediated transcriptional activation (Johansen and Prywes, 1993; Lee et al., 1992). Western blotting analysis illustrated high-level expression (up to 50-fold increase) for both wild-type and mutant SRF proteins (Fig. 4, middle panel). Secondary SRF protein bands associated with SRF overexpression were most likely caused by cellular protease activities generating proteolytic SRF subfragments.
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MTT assays revealed that DM5 (lacking the activation domain) failed to elicit cell death as expected (Fig. 4, bottom panel). Interestingly, DM1 (lacking alanine- and glutamate-rich domains) also failed to induce cell death, suggesting the involvement of multiple SRF protein domains outside its DNA-binding domain in cell killing (Fig. 4, bottom panel). Consistent with the notion that transcriptional squelching does not require DNA-protein interaction, DM3 (lacking the DNA-binding domain) still retained its cytotoxicity, although at a reduced potency compared to the wild-type SRF. Thus, an intact DNA-binding domain of SRF appears dispensable for manifestation of cellular toxicity. Further supporting this notion of coupled transcriptional squelching and cell killing is the additional demonstration that DM3, like wild type (WT)-SRF, remained capable of transcriptional squelching (Fig. 5). DM1 and DM5, on the other hand, failed to exhibit transcriptional squelching along with their abolished cytotoxic effect.
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Minor Role of Caspase and Calpain in Transcriptional Squelching–Mediated Cytotoxicity
Cell death can be executed through caspase and/or calpain proteolytic pathways (Cohen, 1997
; Kositprapa et al., 2000; Squier et al., 1994). To examine the mechanism of transcriptional squelching–mediated cytotoxicity, we targeted several components of the caspase/calpain proteolytic cascades. The porcine bcl-xL cDNA, encoding an antiapoptotic protein of the bcl-2 family (Hockenbery et al., 1990), was cloned (GenBank accession #AF216205), expressed by the adenoviral vector, and tested for its effect on squelching-mediated cell death. Also examined was IC (Kositprapa et al., 2000; Mampuru et al., 1996). Figure 6 (right panel) reveals a slight but statistically significant attenuation of cell death by Bcl-xL in the double expressors. However, overexpression of calpastatin exacerbated squelching-induced cell death in the corresponding double expressors. The slight protective effect of Bcl-xL prompted us to examine the effect of the pan caspase peptide inhibitor zVAD (Ekert et al., 1999). We previously demonstrated that the zVAD caspase inhibitor was effective in blocking p53-mediated cell death (Kositprapa et al., 2000). Again, this pan caspase inhibitor only marginally attenuated cell death (Fig. 6, left panel). These results together indicate that transcriptional squelching–induced cell killing proceed largely independent of the caspase or calpain proteolytic pathways.
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Serum Requirement and Cell-Type Sensitivity
Since serum withdrawal is required for cell death triggered by overexpression of c-myc (Evan et al., 1992
; Prendergast, 1999), we further examined the influence of serum withdrawal on transcriptional squelching–mediated cell death. Cells were plated in culture media containing 5%, 0.5%, and 0% FBS after adenoviral infection, and cell viability and cell death were assessed by MTT and LDH release assays, respectively. Figure 7 (top panel) shows that cell death as assessed by LDH release was increased by SRF expression regardless of the serum concentrations, indicating that, in contrast to c-myc–mediated cell death, SRF-induced cell killing can be initiated without serum withdrawal. Consistent with the LDH assays, parallel MTT assays (Fig. 7, bottom panel) revealed SRF-mediated cell loss in all three culture medium conditions. We further compared in parallel cell-killing potency of SRF and the tumor suppressor protein p53, a well-known apoptotic inducer, in the context of multiple cell types. Both primary cells (MSCs and cardiomyocytes) and immortalized cells (Sol8 myoblasts, P19 embryonal carcinoma cells, and HIIEC3 hepatoma cells) were used for the comparison using the same adenoviral expression system. Figure 8 (top panel) revealed that SRF cytotoxicity was observed in all cell types except HIIEC3 hepatoma cells and that cardiomyocytes appeared to be most sensitive. In comparison, only MSCs and cardiomyocytes were sensitive to p53 cytotoxicity. Western blotting (Fig. 8, bottom panel) revealed that the resistance of HIIEC3 hepatoma cells to SRF cytotoxicity might be due to a lower SRF expression level in these cells compared to other cell types. Thus, SRF-mediated transcriptional squelching appears to elicit a more potent cell-killing effect than overexpression of p53.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current study documents a hitherto unrecognized mode of cell killing caused by SRF-mediated transcriptional squelching. Although the transcriptional squelching phenomenon was documented previously by others and us (Lee et al., 1992
, 1998; Prywes and Zhu, 1992), its effect on cell viability has been overlooked due to the wide use of transient transfection and in vitro transcription assays. It should be noted that although expression of SRF can be induced in various cellular processes, the extent of induction appears relatively mild and can be short lived (Browning et al., 1998; Lee et al., 1992; Soulez et al., 1996; Spencer and Misra, 1999). The use of the adenoviral expression system driven by the potent CMV promoter allows us to artificially achieve up to a 50-fold increase in SRF protein levels, and the outcome of this sustained SRF induction appears to be robust cell killing affecting multiple cell types including normal and abnormal proliferating cells as well as postmitotic cells such as cardiomyocytes. That cardiomyocytes appear more sensitive to the SRF cytotoxicity as shown here may explain the previously unexplained demonstration that cardiac-restricted high-level expression of SRF caused severe cardiomyopathy in mice (Zhang et al., 2001a). Notably, the highly conserved DNA-binding domain of SRF was disrupted in our DM3 mutant, and yet, this SRF mutant retained its ability to induce cell death. Consistent with this finding again, cardiac-specific expression of an SRF mutant deficient in DNA-binding activity also caused cardiomyopathy (Zhang et al., 2001b).
We conclude that SRF-mediated transcriptional squelching induces potent cell killing by derailing the endogenous transcription machinery independent of promoter DNA–binding events. This squelching phenomenon is associated with introduction of high levels of a potent transcriptional activator into eukaryotic cells and is thought to result from titration of one or more essential transcription factors present in limiting amounts (Gill and Ptashne, 1988; Natesan et al., 1997). Along this line, we showed that adenovirus-expressed SRF suppressed rather than activated the activities of the c-fos promoter, and high levels of SRF could repress gene promoters independent of the promoter SRE/CArG element. The DNA-binding domain of SRF is not absolutely required for and multiple SRF protein domains are involved in cell killing. Interestingly, a proline- and glutamine-rich protein was found to promote neuronal cell death (Gomes et al., 1999). The SRF DM5 mutant lacks the C-terminal transcriptional activation domain, which is also rich in proline and glutamine residues, concurrent with its greatly attenuated cell-killing effect. This transactivation domain of SRF has been shown to physically and functionally interact with the large subunit of TFIIF (Zhu et al., 1994). We note that p53-mediated squelching has been documented and could be reversed by either excess TFIIB or TFIID (Liu and Berk, 1995). It would be of interest to determine whether high levels of TFIIF might attenuate cell killing caused by SRF-mediated squelching.
Aside from the basal transcription machinery, numerous sequence-specific transcription factors have been shown to interact with SRF (Chai and Tarnawski, 2002; Miano, 2003). The SRF DM1 mutant, failing to kill cells at high-level expression, lacks an N-terminal domain containing tracts of consecutive alanine and glutamate residues, which can represent a unique structural target for protein interactions. The absence of this unique structural domain might weaken SRF-mediated protein interactions, thus also attenuating cell killing. Since the MADS box is sufficient for SRF protein dimerization, DNA binding, and recruitment of ternary complex factors (Norman et al., 1988), we suggest that transcriptional squelching of ternary complex factors such as p62TCF and myocardin is unlikely to be involved in cell killing. Consistent with this notion, we found that cell death proceeded unabated when SRF and p62TCF were coexpressed in the cell.
Experimental modulation of intracellular SRF levels appears to bear therapeutic implications. For instance, we note that depletion of cardiac SRF by overexpression of antisense SRF sequences could improve the cardiac performance in transgenic mice (Chai and Tarnawski, 2002). Depletion of SRF by RNA interference could enhance the proliferation and migration rates of vascular smooth muscle cells (Kaplan-Albuquerque et al., 2005). Whether these observed beneficial effects of reduced SRF levels stem from thwarted transcriptional squelching remains unclear. On the other hand, artificially elevated SRF levels causing massive transcriptional squelching could represent a feasible module for killing cancer cells as demonstrated here. Unlike c-myc–induced cell death, which requires serum withdrawal (Evan et al., 1992), squelching-induced cell death does not require serum withdrawal. Unlike p53-induced cell death, squelching-induced cell death does not affect expression of bcl-2 (data not shown) and appears more potent than that mediated by p53 overexpression. Squelching-mediated cell killing may be largely necrotic in nature since it could not be effectively intervened by blocking the caspase/calpain proteolytic pathways or fine-tuning the bcl-2 rheostat as shown here. Multiple genes have been explored for killing tumor cells including tumor-suppressing genes, proapoptotic genes, and antiangiogenic genes (Cory and Adams, 2005; Kaplan, 2005). We suggest that manipulation of intracellular SRF protein levels may be used as a cell-killing module mediated by transcriptional squelching.
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ACKNOWLEDGMENTS |
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We would like to thank Gail Willsky for providing HIIEC3 hepatoma cells, Kirk Hammond for providing Ad-LacZ, Bert Vogelstein for providing Ad-p53, and Richard Salvi for providing Ad-Hath1. This work was supported by American Heart Association and Biomedical Research Service Center of University at Buffalo.
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