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Molecular and Cellular Biology, June 2007, p. 3920-3935, Vol. 27, No. 11
0270-7306/07/$08.00+0     doi:10.1128/MCB.01219-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Upregulation of Twist-1 by NF-B Blocks Cytotoxicity Induced by Chemotherapeutic Drugs

Can G. Pham,^1,2 Concetta Bubici,² Francesca Zazzeroni,^2, James R. Knabb,² Salvatore Papa,² Christian Kuntzen,² and Guido Franzoso^2*

Department of Pathology,¹ The Ben May Institute for Cancer Research, The University of Chicago, 924 East 57th Street, Chicago, Illinois 60637²

Received 6 July 2006/ Returned for modification 5 September 2006/ Accepted 19 February 2007

	ABSTRACT

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NF-B/Rel transcription factors are central to controlling programmed cell death (PCD). Activation of NF-B blocks PCD induced by numerous triggers, including ligand engagement of tumor necrosis factor receptor (TNF-R) family receptors. The protective activity of NF-B is also crucial for oncogenesis and cancer chemoresistance. Downstream of TNF-Rs, this activity of NF-B has been linked to the suppression of reactive oxygen species and the c-Jun-N-terminal-kinase (JNK) cascade. The mechanism by which NF-B inhibits PCD triggered by chemotherapeutic drugs, however, remains poorly understood. To understand this mechanism, we sought to identify unrecognized protective genes that are regulated by NF-B. Using an unbiased screen, we identified the basic-helix-loop-helix factor Twist-1 as a new mediator of the protective function of NF-B. Twist-1 is an evolutionarily conserved target of NF-B, blocks PCD induced by chemotherapeutic drugs and TNF- in NF-B-deficient cells, and is essential to counter this PCD in cancer cells. The protective activity of Twist-1 seemingly halts PCD independently of interference with cytotoxic JNK, p53, and p19^ARF signaling, suggesting that it mediates a novel protective mechanism activated by NF-B. Indeed, our data indicate that this activity involves a control of inhibitory Bcl-2 phosphorylation. The data also suggest that Twist-1 and -2 play an important role in NF-B-dependent chemoresistance.

	INTRODUCTION

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Nuclear factor-B/Rel (NF-B/Rel) transcription factors play a central role in controlling programmed cell death (PCD) (reviewed in reference 8), a fundamental process for physiological elimination of a cell (9, 19). Members of this so-called Rel family of proteins, which in mammals include RelA (p65), RelB, Rel (c-Rel), p50/105 (NF-B1), and p52/100 (NF-B2), are expressed in virtually all tissues and can form almost all possible combinations of homo- and heterodimeric, DNA-binding complexes—the most abundant of which is the RelA-p50 heterodimer (8, 43). In cells, these NF-B complexes can rapidly be activated by a spectrum of stimuli that ultimately cause their translocation from the cytoplasm to the nucleus, where they induce the transcription of coordinate arrays of target genes, including those regulating inflammation, immunity, and cell survival (8, 43).

The activation of NF-B blocks PCD induced by numerous cytotoxic triggers, including chemotherapeutic drugs, ionizing radiation, and ligand engagement of death receptors, such as those of the tumor necrosis factor receptor (TNF-R) family (8, 27, 32). Knockout and other studies have shown that this prosurvival activity of NF-B plays an obligatory role in organogenesis, lymphopoiesis, and inflammation, as well as in homeostasis and the function of the liver, skin, and central nervous system (8). RelA-deficient embryos die in utero from massive liver apoptosis, and mouse embryonic fibroblasts derived from these embryos exhibit marked sensitivity to TNF-R-induced PCD (8, 32).

Notably, an inappropriate antagonism of PCD by NF-B is a key pathogenetic element in prevalent human diseases, and this has profound implications for the treatment of these diseases (8, 17, 18, 27, 35). The prosurvival activity of NF-B is crucial for oncogenesis and chemoresistance in cancer (17, 18, 27), and a growing list of human malignancies is now being successfully treated with blockers of NF-B, such as proteasome inhibitors (27, 32). Global blockers of NF-B, however, have severe side effects, including immunosuppressive effects, which greatly limit their clinical use (27). A preferable approach to treatment of these malignancies would be, therefore, to block select downstream targets of NF-B, rather than NF-B itself. These targets, however, remain for the most part unknown. Consequently, the mechanisms through which NF-B orchestrates the inhibition of PCD in cancer cells are poorly understood.

In the case of TNF-Rs, the basis for the protective activity of NF-B is now beginning to be unveiled. The fact that cytotoxicity induced by TNF- involves an accumulation of reactive oxygen species (ROS) (14, 36, 40, 52) and an induction of persistent activation of the c-Jun-N-terminal-kinase (JNK) mitogen-activated protein kinase cascade (7, 8, 32, 49) recently emerged. Indeed, ROS and JNK activities can trigger both the caspase-dependent (i.e., apoptotic) (36, 54) and caspase-independent, necrotic (14, 40, 52, 54) pathways of PCD. Several studies now indicate that a program of gene expression induced by NF-B counters both this accumulation of ROS (36, 40) and this activation of JNK signaling downstream of TNF-Rs (7, 32, 49, 52). The antioxidant action of NF-B is believed in fact to represent an additional, indirect mechanism by which NF-B promotes a restraint of JNK activation, as this activation of JNK by TNF- depends on ROS (14, 32, 36, 40). The NF-B-regulated containment of ROS is mediated by a distinct subset of NF-B target genes, including ferritin heavy chain and manganous superoxide dismutase (14, 32, 36). Yet, it is now clear that the program for cell survival that is activated by NF-B has both tissue- and context-specific components (8, 32), and so, it is uncertain whether the inhibition of ROS and/or JNK signaling also plays a role in NF-B-mediated chemoresistance in cancer.

To understand the basis for the prosurvival activity of NF-B in cancer and unravel an additional mechanism(s) by which NF-B controls TNF--induced killing, we sought to identify unrecognized protective genes that are regulated by NF-B. Using an unbiased gene chip screen, here we identify Twist-1 as a cDNA capable of protecting RelA^–/– cells from PCD elicited by either TNF- or chemotherapeutic drugs. Twist-1 is a so-called basic-helix-loop-helix transcription factor and a well-characterized downstream target of NF-B (5, 16, 44, 45, 48, 55). In Drosophila melanogaster, Twist was previously shown to be under the transcriptional control of Dorsal (13, 30, 50), a homolog of NF-B, and to play a key role in dorsal-ventral patterning (4), cooperating with NF-B at promoters of common target genes (12, 34, 42, 45, 47).

This NF-B-mediated control of Twist genes is conserved in mammalian cells (16, 44, 45, 48, 55). Here we found that in these cells, NF-B is even sufficient alone for the transcriptional upregulation of these genes. We also found that the protective activity of Twist-1 is independent of an interaction with NF-B dimers and that it does not involve an inhibition of the JNK pathway. Notably, this protective activity of Twist-1 is capable of blocking both the apoptotic and the necrotic pathway of PCD activated by chemotherapeutic agents. Other studies have indicated that Twist-1 is expressed at high levels in certain cancers (20, 22, 25, 26, 37, 38, 51, 58) and that this expression of Twist-1 in cancer is essential to counter cytotoxicity induced by these agents (20, 56). All together, our data show that the upregulation of Twist-1 (and Twist-2) represents a novel mechanism by which NF-B antagonizes PCD in cancer, a mechanism that seemingly acts downstream of the JNK cascade and is independent of an interference with the p53 and p19^ARF tumor suppressor pathways. Our data also indicate that the protective action of Twist-1/2 involves a suppression of inhibitory phosphorylation of Bcl-2 on Ser-87. Hence, Twist factors may represent suitable new targets for anticancer therapy.

	MATERIALS AND METHODS

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Library construction and selection, microarray analyses, RT-PCR, and Northern blotting. Detailed information on library preparation, its selection through the death trap screen, and the gene chip analysis can be found in references 7 and 36. For reverse transcriptase PCR (RT-PCR), total RNA was prepared from the HtTA-1, HtTA-RelA, CCR43, and PC-3 cell lines using TRIzol (Invitrogen), and the first-strand cDNA was synthesized using the Superscript first-strand synthesis system (Invitrogen) and 1 µg of RNA as the template, according to the manufacturer's instruction. For DNA amplification, we used an annealing temperature of 62°C and the following primer sets: for human Twist-1, 5'-CAGGGCCGGAGACCTAGATGTCATTG-3' (sense; primer a) and 5'-GCACGACCTCTTGAGAATGCATGCATG-3' (antisense; primer b); 5'-CAGAGCGACGAGCTGGACTCCAAGAT-3' (sense; primer c) and 5'-TGCCGTCTGCCACCTGAGAGGCGAAG-3' (antisense; primer d); and for human RelA, 5'-CCCGGACCGCTGCATCCACAGTTTCC-3' (sense) and 5'-CCACTTGTCGGTGCACATCAGCTTGCG-3' (antisense). Primer sequences for ß-actin were described previously (59). To further ensure specificity, in Fig. 1, Twist-1 PCR products were transferred by Southern blotting onto nitrocellulose membranes, and these were then probed with the antisense oligonucleotide labeled with ³²P by T4 kinase reaction. Northern blotting was performed as detailed elsewhere, using ³²P-labeled probes prepared from mouse Twist-1, IB, or GAPDH cDNAs (59).

View larger version (25K): [in this window] [in a new window]   FIG. 1. The activation of NF-B complexes is sufficient for the transcriptional upregulation of Twist-1. (A) Western blots showing kinetics of induction of RelA and c-Rel in the HtTA-RelA and CCR43 cell lines, respectively, following the removal of tetracycline from the culture medium. As expected, the induction of RelA led to the upregulation of c-Rel, a known target of NF-B (8). ß-Actin is shown as the loading control. (B) RT-PCR showing the induction of mature (spliced) (top) and immature (unspliced) (middle) Twist-1 transcripts following the activation of RelA or c-Rel in HtTA-RelA and CCR43 cells, respectively, but not in control cells (HtTA-1). The PCR primers used are indicated in panel C. Times reflect the duration of the withdrawal of tetracycline prior to the extraction of the total RNA. No amplification signal was detected when samples from mock RT reactions were used as templates for PCR (data not shown) if any DNA contamination of the RNA samples is excluded. Southern blotting was performed using 32P-labeled probes specific for Twist-1 mRNAs (top and middle). Also shown as loading controls are the ethidium bromide-stained ß-actin products. (C) Schematic representation of the relative localizations within the Twist-1 locus of PCR primers a and b and of primers c and d, used for the detection of spliced and unspliced Twist-1 transcripts, respectively. UTR, untranscribed region.

The bicistronic, murine stem cell virus-based retroviral vector MIGR1, expressing enhanced green fluorescent protein (eGFP), has been described previously (59). MIGR1-Twist-1-Flag and MIGR1-Twist-2-Flag were constructed by excising the Twist-1-Flag and Twist-2-Flag cDNA-containing inserts, respectively, from the corresponding pcDNA3.1-Twist-Flag plasmids with PmeI and ligating them into the XhoI site of MIGR1, filled in with the Klenow fragment. For generation of MIGR1-CTwist-2, the truncated murine CTwist-2 cDNA was amplified by PCR from pcDNA3.1-CTwist-2 (a kind gift of D. Sosic and E. N. Olson [45]) using the following primers: 5'-GGAAGATCTGCCACCATGGAACAAAAGCTGATTTCT-3' (sense) and 5'-GGAGAATTCCTAGTAGAGGAAGTCTATGTACCTGGC-3' (antisense) (internal BglII and EcoRI restriction sites are underlined; starting ATG and stop TAG codons are in bold). The resulting PCR product was then digested with BglII and EcoRI and ligated into the same DNA restriction sites of MIGR1. The lentiviral vector pLentiLox3.7 (pLL), encoding eGFP, and the pLL-shRelA and pLL-shMut-3 vectors, expressing RelA-specific and nonspecific short-hairpin RNA (shRNA) oligonucleotides, respectively, were described previously (57). To create pLL-shTwist-1, expressing shRNAs targeting human Twist-1, the following DNA oligonucleotide linker was ligated into the HpaI and XhoI restriction sites of pLL: 5'-tAAGCTGAGCAAGATTCAGAttcaagagaTCTGAATCTTGCTCAGCTTtttttttc-3' (sense only; the 19-nucleotide target sequence is in uppercase letters). All clonings were confirmed via appropriate restriction digestions and nucleotide sequencing.

MIGR1 retroviral preparations in Phoenix cells and retroviral transductions of fibroblasts were performed as described previously (59). The preparation of pLL lentiviruses in 293T cells and transduction of human prostate adenocarcinoma PC-3 cells were also carried out essentially as detailed previously (57). Briefly, these cells were seeded at 10⁵ cells/well in six-well plates and, 24 h later, incubated with high-titer lentiviral preparations for 20 h at 37°C in the presence of Polybrene (5 µg/ml). Cultures were then washed twice with complete medium, and infection efficiency (i.e., the percentage of eGFP⁺ cells) was monitored after an additional 24 h by flow cytometry (FCM). Immortalized RelA^–/– fibroblasts, 3DO-IBM T-cell hybridoma clones stably expressing IBM, and the HeLa-derived cell lines HtTA-1, HtTA-RelA, and CCR43 were cultured as detailed before (33, 36). PC-3 cells and p19^ARF–/– fibroblasts (kindly provided by M. Peter and C. Sherr, respectively) were maintained in RPMI 1640 and Dulbecco's modified Eagle's medium (Invitrogen), respectively, each supplemented with 10% fetal calf serum, penicillin, and streptomycin. The 3DO-IBM-Twist-1 and control 3DO-IBM-Hygro clones, stably expressing Twist-1-Flag and harboring empty pcDNA-(Hygro)-Flag plasmids, respectively, were established through the electroporation of appropriate linearized pcDNA3.1-Twist-1-Flag or pcDNA3.1-(Hygro)-Flag plasmids into 3DO-IBM clone 25 (described in reference 7), followed by limiting dilutions in 96-well plates and double selection with neomycin (1 mg/ml) (Cellgro) and hygromycin (450 U/ml) (Calbiochem).

Death assays, light microscopy, and transmission electron microscopy (TEM). For viability and death assays, RelA^–/– fibroblasts were seeded onto 60-mm dishes, 48-well plates, or 96-well plates at a density of 0.5 x 10⁶/dish, 0.3 x 10⁵/well, or 0.1 x10⁵/well, respectively, and 24 h later, they were treated with recombinant murine TNF- (Preprotech, Rocky Hill, NJ) or daunorubicin (Sigma), as indicated in the figure legends. For viability assays, p19^ARF–/– fibroblasts were seeded at a density of 0.4 x 10⁶/dish onto 60-mm dishes and then treated as described above. Cell viability was determined at the times indicated in the figures by manual counting of adherent cells; propidium iodide (PI) nuclear staining of pooled, detached, and adherent cells; metabolic tetrazolium salt (MTS) assays (CellTiter96AQ; Promega); or light microscopy, as appropriate. PI nuclear staining assays were performed as described previously (7, 36, 59). MTS assays were carried out according to the manufacturer's instructions. Light microscopic images were acquired using an Axiovert S-100 microscope (Zeiss), a 10x objective, and appropriate Zeiss software. Cell death assays was performed by using a cell death detection enzyme-linked immunosorbent assay (ELISA) kit (ELISA^PLUS; Roche Diagnostic Corporation, Indianapolis, IN), according to manufacturer's instructions as described previously (33, 36).

With PC-3 lines expressing Twist-1-specific or control shRNAs, cells were seeded onto 12-well plates or 48-well plates at a density of 0.4 x 10⁶ cells/well or 0.3 x 10⁵/well, respectively, and, 24 h later, exposed to the daunorubicin analogue VP-16 (Sigma) or cisplatin (provided by the CAM Outpatient Chemotherapy Pharmacy at the University of Chicago). Cell viability was then monitored at the times indicated in the figures by counting of adherent cells or performing PI nuclear staining or light microscopy, as indicated. Cell death was measured by using the cell death detection ELISA^PLUS kit (33, 36).

For TEM, treated and untreated cells were detached by trypsin, pelleted by centrifugation, washed twice in serum-free medium, and then fixed for 30 min using 4% paraformaldehyde, 1.25% glutaraldehyde, 0.1 M sodium cacodylate. Secondary fixation was achieved using a buffer containing 1% OsO₄ and 0.1 M sodium cacodylate buffer. Fixed cells were then dehydrated with ethanol, stained using uranyl and lead acetate, and, finally, embedded in Epon. Sections (50 nm thick) were analyzed using an FEI Tecnai F30ST electron microscope at the University of Chicago Electron Microscopy Facility. Microscope settings that were used are as follows: emission and accelerating voltages were 4,200 V and 300 kV, respectively, the C2 aperture was 150 µm, and the objective aperture was 30 µm. Images were acquired using a Gatan (model 4Kx4K) charge-coupled-device camera.

Western blots, JNK kinase assays, mitochondrial depolarization, FCM, and reagents. Cell extracts were prepared in Triton X-100 lysis buffer as described previously (59). Protein concentrations were determined via standard colorimetric assays (Bio-Rad), and equal amounts of proteins were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose. Immunodetection procedures were carried out using enhanced chemiluminescence, as detailed previously (33, 59). The following primary antibodies were used: anti-RelA and anti-c-Flip_L (Stressgen, Victoria, BC, Canada); anti-Flag M2 (Sigma); anti-p21 (F-5), anti-MDM2 (SMP14), anti-RIP-1, and anti-JNK (BD Biosciences Pharmingen); anti-c-Rel, anti-Fas-associated death domain (FADD), anti-Bcl-2, anti-phospho-Bcl-2 (Ser-87), anti-Bad, anti-Bax (N-20), and anti-ß-actin (Santa Cruz); anti-p53 (Oncogene); anti-caspase-3 (Cell Signaling); anti-Bid (R&D); and anti-caspase-8 (C-15; a kind gift from M. Peter, The University of Chicago). Mitochondrial depolarization and JNK kinase assays were performed as described previously (33, 59). FCM for the detection of the surface expression of TNF-R1 was carried out by standard procedures. Biotinylated, monoclonal anti-TNF-R1 and isotype-matched control antibodies and streptavidin-allophycocyanin (APC) were purchased from HyCult Biotechnology, BD Biosciences Pharmingen, and Molecular Probes, respectively. Freshly stained samples were read using a FACSCanto apparatus, which collected 20,000 events, and analyses were preformed using the FlowJo software. The antioxidant butylated hydroxyanisole (BHA), the pan-caspase inhibitor zVAD (N-benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethylketone), and the JNK inhibitor SP600125 were from Sigma, MP Biomedicals, and Calbiochem, respectively.

	RESULTS

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Isolation of Twist-1 from libraries enriched in protective genes. To understand the mechanisms by which NF-B blocks TNF--induced PCD and promotes chemoresistance in cancer, we sought to identify genes that possess cytoprotective activity and are regulated by NF-B. To this end, we used gene microarrays in a systematic screen of cDNA libraries that had been enriched in protective plasmids through selection with TNF- in RelA^–/– fibroblasts (details on library construction and selection and the gene chip screen can be found in references 7 and 36). By in vitro transcription, probes were prepared from the original and selected libraries and used to interrogate Affymetrix Mu6500 microchips (36). Putative protective cDNAs were defined as those that increased in frequency during selection and so yielded stronger hybridization signals with probes prepared from the enriched library than with those prepared from the original library (7, 36). Since this approach required no information about gene sequence and/or gene function, it provided an unbiased method for isolating genes capable of attenuating TNF--induced PCD in NF-B-deficient cells (7, 36). It also enabled ranking of these genes according to their signal log ratio (SLR) scores, which correlate with their extent of enrichment during selection and, hence, provides a semiquantitative indication of their antiapoptotic efficacies (36). Of the genes represented in the Mu6500 chip, 90 exhibited SLR values higher than 1 (36; data not shown). Validating our approach, two of the highest SLR scores in this system were assigned to cDNAs encoding RelA and dominant negative FADD (36), two well-characterized blockers of TNF--induced killing (8, 54). Using this approach, we previously identified the ferritin heavy chain as a new mediator of the antioxidant and protective activities of NF-B (36).

Another cDNA that was highly enriched by selection in RelA^–/– cells was found to encode Twist-1 (exhibiting an SLR score of 2.0 [36]). Further analyses confirmed the presence of full-length Twist-1 cDNAs in the selected library (data not shown), suggesting that these cDNAs are bona fide inhibitors of PCD. Interestingly, this library also contained plasmids encoding the closely related factor, Twist-2 (also known as Dermo-1) (4, 37, 45). These plasmids, however, were enriched to a lesser extent than those encoding Twist-1, having an SLR score of 1.0. Twist genes are evolutionarily conserved transcriptional targets and mediators of several biological functions of NF-B (13, 16, 30, 45, 48, 50, 55). Yet, isolation of these genes in our screen in RelA null cells was somewhat surprising since, to carry out function, Twist proteins are believed to require functional NF-B dimers (34, 42, 45) (discussed below).

Twist-1 is a downstream target of NF-B. Twist-1 is a known target of NF-B, and its dependence on NF-B for induction by cytokines and developmental cues is well established in various systems (13, 16, 30, 45, 48, 50; see also below). Thus, we sought to determine whether, in addition to being required, NF-B was sufficient for the transcriptional activation of Twist-1. To this end, we used the HeLa-derived cell lines HtTA-RelA and CCR43, where the expression of RelA and Rel, respectively, can be induced by the withdrawal of tetracycline from the culture medium and so in the absence of extracellular stimulation (Fig. 1A) (36). As shown in Fig. 1B (top), following the removal of tetracycline, mature Twist-1 transcripts accumulated markedly in HtTA-RelA and CCR43 cells but not in control HtTA-1 cells, suggesting that, at least in HeLa cells, the nuclear translocation of either RelA or Rel is sufficient alone to upregulate Twist-1 expression. As expected, NF-B-mediated upregulation of Twist-1 mRNAs involved a transcriptional event, as immature (unspliced) transcripts were also induced upon the conditional expression of RelA or Rel (Fig. 1B, middle, and C).

Twist-1 attenuates TNF--induced PCD in NF-B-deficient cells. To ensure that the upregulation of Twist-1 mediates a protective function, we examined the effect of Twist-1 expression on TNF--induced PCD in RelA^–/– cells. MIGR1 retroviruses encoding Flag-tagged, full-length Twist-1 or empty MIGR1 controls were introduced into these cells and levels of ectopically expressed Twist-1 and MIGR1-encoded eGFP were assessed by using anti-Flag immunodetection and FCM (Fig. 2A, right, and data not shown, respectively). Due to their survival defect, RelA^–/– fibroblasts succumb rapidly upon exposure to TNF- (MIGR1 [36]). Strikingly, however, the expression of Twist-1 in these cells conferred strong protection against TNF--induced PCD (Fig. 2A, left; MIGR1-Twist-1 bars). Similar results were obtained using a quantitative, ELISA for monitoring cell death (Fig. 2B) (33, 36). This Twist-1-mediated cyto-resistance was most pronounced at early times (6 and 8 h) (Fig. 2A), suggesting that, in accordance with previous studies, additional factors must account for the long-lasting, complete protection normally afforded by NF-B (7, 8, 32, 54). A similar protective activity was observed with Twist-2 (data not shown) although, consistent with the lower SRL value assigned to Twist-2 cDNAs, in RelA null cells this activity was somewhat weaker than that of Twist-1.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Twist-1 attenuates TNF--induced PCD in NF-B-deficient cells. (A) The ectopic expression of Twist-1 rescues RelA null fibroblasts from TNF--induced PCD. MIGR1- and MIGR1-Twist-1-transduced-RelA–/– cells were seeded onto 60-mm dishes at a density of 0.5 x 106 cells/dish and, 24 h later, treated either with cycloheximide (CHX) alone (0.1 µg/ml) or with CHX (0.1 µg/ml) plus TNF- (100 U/ml). At the times indicated, cell viability was assessed by determining total cell numbers in both TNF--treated and control cultures. For this, adherent cells were recovered by trypsinization followed by centrifugation, resuspended in serum-containing medium, and counted by hemacytometry (36). Fractions of GFP+ cells were assessed by FCM. Values reflect the percentages of live (i.e., adherent) GFP+ cells (determined by combining cell counting and FCM) relative to the numbers observed in cultures treated with CHX alone (left). These percentages represent means ± standard deviations from three independent experiments. Levels of ectopic Flag-Twist were monitored by Western blotting (right). (B) ELISAs of PCD confirming the protective activity of ectopic Twist-1 in RelA null cells. Cell death was monitored in the same MIGR1- and MIGR1-Twist-1 RelA-deficient lines used to obtain the panel A results, following treatment with CHX alone (0.1 µg/ml) or together with a low (100 U/ml) or high (250 U/ml) concentration of TNF-, as indicated. Treatment times are also shown. Assays were preformed as described previously (33, 36), and cell death is expressed as arbitrary units. Values represent means ± standard deviations from three independent experiments. (C) Survival of representative Hygro- and Twist-1-expressing, 3DO-IBM clones (IBM-Hygro and IBM-Twist-1, respectively) after treatment with TNF-. For survival assays, cells were plated onto 24-well dishes at 3.5 x 105 cells/well and then exposed to TNF- (15 U/ml) or left untreated. Viability was assessed at 6 h by performing PI nuclear staining assays. DNA content was determined with FCM analysis and analyzed using the FlowJo software. Percentages of survival reflect fractions of live cells (i.e., of at least G1 DNA content) in the TNF--treated cultures relative to the number of live cells in the untreated cultures (top). Clone numbers are shown. Levels of expression of Twist-1, IB, and GAPDH mRNAs in each clone were monitored by Northern blotting (bottom).

Twist-1 blunts TNF--induced apoptotic signaling. TNF- has the ability to trigger both the apoptotic and the necrotic pathway of PCD (9, 14, 19, 36, 40, 52, 54). We found, however, that in RelA^–/– fibroblasts and 3DO-IBM clones, TNF--induced killing is almost completely blocked by treatment with the pan-caspase inhibitor zVAD-_fmk (36), suggesting that in these cells, such killing relies mainly on an apoptotic mechanism. Thus, to begin to understand how Twist-1 blocks TNF-R-triggered PCD, we monitored its effects on the activation of caspase proteases. In MIGR1-transduced RelA^–/– cells, TNF- elicited progressive depletion of the initiator procaspase-8 (8, 19, 32, 54), beginning as early as 6 h, as shown by Western blotting; see levels of p57 (Fig. 3A). This depletion of procaspase-8 coincided with the proteolysis of the Bcl-2-like factor and caspase-8 substrate Bid (8, 19, 32, 54), as well as of the effector procaspase-3, two unequivocal signs of the activation of caspase-8 (8, 19, 32, 54). As expected, the cleavage of Bid and procaspase-3 was accompanied by an accumulation of their active products, tBid and p17/p12, respectively, beginning by 4 to 6 h (Fig. 3A). Remarkably, in RelA null cells, these events were markedly delayed by MIGR1-Twist-1 (Fig. 3A). Ectopic expression of Twist-1 also blocked caspase-induced DNA fragmentation (Fig. 3B; see sub-G₁ pools), another hallmark of apoptosis (9, 19, 54). This fragmentation was instead readily observed in MIGR1-transduced RelA-deficient cells, as demonstrated by PI nuclear staining assays (Fig. 3B). Of note, however, unlike what was seen with MIGR1-RelA (36; data not shown) and consistent with the data shown in Fig. 2A, the inhibitory effects of Twist-1 on TNF--induced caspase activation and DNA fragmentation were incomplete. With time, in fact, this caspase activation became apparent also in cells expressing Twist-1 (Fig. 3A, MIGR1-Twist-1 lanes at 10 and 12 h).

View larger version (45K): [in this window] [in a new window]   FIG. 3. Twist-1 antagonizes apoptotic signaling induced by TNF- in RelA–/– cells. (A) Western blots showing that Twist-1 is capable of suppressing TNF--induced caspase activation and Bid processing. MIGR1- and MIGR1-Twist-1-transduced RelA–/– cells were seeded as described for Fig. 2A and stimulated with TNF- (100 U/ml) plus CHX (0.1 µg/ml). At the times indicated, extracts were prepared from TNF--treated and control cultures (i.e., 0-hour time points) and used to assess the kinetics of activation of caspase-8 (p57), caspase-3, and Bid as determined by immunoblotting. Also indicated are the levels of ectopically expressed Twist-1. ß-Actin is shown as the loading control. Antibodies and intact and cleaved polypeptide products are indicated (on the left- and right-hand sides, respectively). (B) PI nuclear staining assays showing that TNF--induced DNA fragmentation in RelA–/– cells is blocked by the ectopic expression of Twist-1. FCM histogram profiles showing DNA content in MIGR1- and MIGR1-Twist-1-transduced RelA–/– cells which were either left untreated (i.e., CHX alone) (unstimulated [US] panels) or treated with TNF- (100 U/ml) plus CHX (0.1 µg/ml) (TNF panels), as indicated. Cells were collected 14 h after these treatments, and DNA fragmentation in these cultures was assessed by performing PI nuclear staining followed by FCM analysis. Values represent percentages of cells manifesting sub-G1 and at least G1 DNA contents, as shown. (C) Twist-1 effectively inhibits MOMP triggered by TNF- in RelA–/– cells. Mitochondrial depolarization in MIGR1- and Twist-1-transduced RelA–/– cultures treated with CHX alone or together with TNF-, as described for panel A, was determined by performing JC-1 fluorescence staining. At 4, 8, 12, and 16 h after TNF- stimulation, detached and adherent cells were harvested, pooled, washed with phosphate-buffered saline (PBS), and then loaded with JC-1 (10 µg/ml in PBS) and incubated at 37°C for 30 min. Cells were then washed three additional times with PBS and analyzed by FCM. Shown are percentages of JC-1+ cells relative to those in cultures treated with CHX alone and reflect means ± standard deviations from three independent experiments. (D) FCM analysis showing comparable levels of surface expression of TNF-R1 in MIGR1- and MIGR1-Twist-1-transduced cells. Cells were stained with a biotin-labeled anti-TNF-R1 antibody (-TNF-R1) (black line) or a biotin-labeled isotype-matched control (anti-immunoglobulin G2a [-IgG2a]) (gray line), as indicated, followed by incubation with streptavidin-APC (SA-APC), and FCM was performed using standard methods. FCM histogram profiles depict APC positivity in MIGR1- and MIGR1-Twist-1-infected RelA–/– cultures and were generated using the FlowJo software as described for Fig. 2C. (E) Western blots showing levels of the indicated proteins in MIGR1- and MIGR1-Twist-1-transduced cells treated with TNF- and CHX or left untreated (0 h-time points) as described for panel A. Extracts were the same as those used to obtain the panel A results. Antibodies are indicated.

View larger version (70K): [in this window] [in a new window]   FIG. 4. Twist-1 blocks both apoptosis and necrosis-like PCD induced by a chemotherapeutic drug. (A) TEM images showing morphological changes in MIGR1- and MIGR1-Twist-1-transduced RelA–/– cells following the induction of PCD by treatment with daunorubicin (5 µM) for 12 and 18 h, as shown. TEM images of representative cells are shown. Treated, control cells (MIGR1) manifest changes consistent with both apoptotic (images b and d) and necrotic (images c and e) PCD. These include chromatin and organelle condensation (characteristic of apoptotic cells) and nuclear disintegration and generalized cell swelling and disorganization (characteristic of necrotic cells) with shells found at late time points (image e). In contrast, daunorubicin-treated RelA–/– cells expressing Twist-1 or Twist-2 (MIGR1-Twist-1 or MIGR1-Twist-2, respectively) by and large retained morphologies similar to those in untreated (UT) cultures (images f, g, and i), even at late times (images h and j), although some signs of apoptosis, such as chromatin condensation, could be observed in some cells at these times (image h). (B) Light microscopic images showing that the pan-caspase inhibitor zVAD-fmk, the antioxidant BHA, and the JNK inhibitor SP600125 can only partially protect MIGR1-transduced RelA–/– cells against cytotoxicity induced by daunorubicin. MIGR1-transduced-RelA–/– cultures were seeded in 60-mm dishes as described for Fig. 2A and then left untreated (–) or treated with zVAD-fmk (50 µM), BHA (100 µM), or SP600125 (50 µM), as shown. Twenty minutes later, where indicated, cells were exposed to daunorubicin (7.5 µM). Control cultures were left untreated (UT). After 16 h of cytotoxic treatment, cell viability was assessed by microscopic inspection, using an Axiovert S-100 Zeiss microscope with a 10x objective. Photos showing these cultures were acquired using Zeiss software. Pretreatments with zVAD, BHA, or SP600125 improved the survival of MIGR1-transduced RelA–/– cells (MIGR1) treated with daunorubicin, compared to that of nonpretreated cells. Also shown is the superior protection afforded by ectopic Twist-1 (MIGR1-Twist-1) against daunorubicin-induced cytotoxicity in RelA–/– cells. (C) Percentages of survival calculated for the cultures depicted in panel B. This viability was extrapolated by normalizing numbers of live cells in daunorubicin-treated cultures to the numbers of live cells in transduced RelA–/– cultures that were not exposed to daunorubicin. -, no pretreatment. Values are means ± standard deviations from three separate measurements. (D) ELISAs of PCD confirming the protective activity of ectopic Twist-1 in RelA null cells. Assays were performed using the same MIGR1- and MIGR1-Twist-1-trasduced, RelA-deficient lines used to obtain the results in panels B and C. Treatment with daunorubicin was at 5 µM, and pretreatments with zVAD-fmk, BHA, and SP600125 were carried out as described for panels B and C, as indicated. Cells were harvested after a 16-hour treatment with daunorubicin, and assays were preformed as described for Fig. 2B. Cell death is expressed as arbitrary units and is shown for both treated and untreated cultures. Values represent means ± standard deviations from three independent experiments. (E) Twist-1 effectively blocks daunorubicin-induced cytotoxicity in RelA–/– cells. Light microscopic images depicting the viability of MIGR1- and MIGR1-Twist-1-transduced RelA–/– cells, seeded as described for Fig. 3A, and left untreated or treated with daunorubicin at a lower concentration than that used for panel B results (i.e., 5 µM) for 15 h. Photos were acquired as described for panel B. (F) MTS assays showing daunorubicin-induced cytotoxicity in MIGR1- and MIGR1-Twist-1-transduced RelA–/– cells. Cells were seeded onto 96-well plates at 104 cells/well and were left untreated or treated with daunorubicin (5 µM) for 15 h. Cultures were then supplemented with 20 µl of CellTiter96AQ reagent (Promega, Madison, WI) and incubated at 37°C for 1 h, and optical densities (O.D.) of the culture supernatants were finally measured using a SpectraMAX 250 microplate spectrophotometer (Molecular Devices) at A490. Values are the differences in optical densities between daunorubicin-treated and untreated cultures (i.e., the optical densities of the untreated cultures minus those of the treated cultures) and represent means ± standard deviations from three independent experiments.

These findings for RelA^–/– cells suggest that, as seen with TNF- (Fig. 2A to C), the ability of Twist-1 and Twist-2 to counter daunorubicin-inflicted killing is independent of an interaction with NF-B dimers. Since RelA^–/– cells retain the expression of NF-B family proteins, such as p50 and c-Rel, it is possible that these proteins can compensate in part for the lack of RelA. To clarify this issue, we tested the activity of the Twist-2 mutant CTwist-2, which fails to interact with NF-B dimers (45). Upon overexpression in RelA^–/– cells, CTwist-2 effectively rescued these cells from daunorubicin-inflicted killing, and its protective efficacy against this killing was comparable to that of wild-type Twist-1 and Twist-2 (Fig. 5A [light microscopy], B [cell counts], C [ELISAs], and D [FCM]). We concluded that Twist factors are potent blockers of cytotoxicity elicited by chemotherapeutic drugs (and TNF-) and that, unlike with other biological functions of these factors, protection against this cytotoxicity does not require an interaction with NF-B dimers.

View larger version (55K): [in this window] [in a new window]   FIG. 5. Twist proteins block daunorubicin-induced PCD through a mechanism that does not involve an interaction with NF-B dimers. (A) Light microscopic images showing that CTwist-2 effectively rescues RelA–/– cells from cytotoxicity induced by daunorubicin. RelA–/– cells were infected with the indicated MIGR1 retrovirues, seeded onto 60-mm dishes as described for Fig. 2A, and then left untreated (UT) or treated with daunorubicin (7.5 µM) as shown. After a 15-hour treatment, cell viability was assessed by microscopic inspection as detailed in the legend to Fig. 4B, using an Axiovert S-100 Zeiss microscope with a 10x objective. Shown are comparable levels of protection afforded by ectopic Twist-1, Twist-2, and CTwist-2 (MIGR1-Twist-1, MIGR1-Twist-2, and MIGR1-CTwist-2 images, respectively) against daunorubicin-induced PCD in RelA–/– cells. (B) Percentages of survival calculated for the cultures depicted in panel A. Viability was extrapolated as described for Fig. 4C by normalizing numbers of live cells in the daunorubicin-treated cultures to the numbers of cells in the corresponding untreated cultures. Values are means ± standard deviations of three indipendent measurements. (C) ELISAs showing PCD in cultures of RelA null cells transduced with MIGR1, MIGR1-Twist-1, MIGR1-Twist-2, or MIGR1-CTwist-2, as shown. Cells were treated with daunorubicin (5 µM) for 16 h or left untreated as indicated. Cell lines were the same those used to obtain the results in panels A and B, and assays were preformed as described in the legend to Fig. 2B. Cell death is expressed as arbitrary units and is shown for both treated and untreated (UT) cultures. Values represent means ± standard deviations from three independent experiments. (D) Comparable levels of infection efficiency by MIGR1 retroviruses of the RelA–/– cultures used to obtain the results shown in panels A to C. FCM histogram profiles showing eGFP positivity in the MIGR1-, MIGR1-Twist-1-, MIGR1-Twist-2-, and MIGR1-CTwist-2-infected RelA–/– cells shown in panels A to C and uninfected RelA–/– controls (No Infection), as indicated. Cells were harvested 48 h after first being exposed to the retroviral preparations, and eGFP positivity in these cultures was assessed by FCM, followed by analysis with the FlowJo software, as described for Fig. 2C. The bar indicates fractions of GFP+ cells.

Twist-1 mediates the NF-B-dependent prosurvival activity in cancer cells.

View larger version (45K): [in this window] [in a new window]   FIG. 6. Twist-1 is required to antagonize cytotoxicity induced by chemotherapeutic drugs in PC-3 prostate cancer cells. (A) RT-PCR showing the downregulation of Twist-1 transcripts following the infection of PC-3 cells with pLL lentiviruses expressing Twist-1-specific, but not control (lane Mut-3), shRNA, as indicated. Shown are ethidium bromide-stained Twist-1 and ß-actin PCR products. Several anti-Twist-1 antibodies were tested for immunoblot detection of endogenous Twist-1 polypeptides and found unsuitable for this purpose (C. Bubici and G. Franzoso, unpublished observations). (B) Percentages of survival of PC-3 cells infected with lentiviruses expressing Twist-1-specific or control (Mut-3) shRNA, as indicated. Cells were seeded onto a 12-well plate at 4 x104 cells/dish and then left untreated or treated with VP-16 (etoposide, 5 µM) for 48 h. Viability was calculated from the same cultures used to obtain the results depicted in panel C, and values were extrapolated as described for Fig. 4C, by normalizing numbers of live cells in the VP-16-treated cultures to the numbers of live cells in the corresponding untreated cultures. Values are means ± standard deviations of three independent measurements. (C) Light microscopic images showing that expression of Twist-1 shRNAs renders PC-3 cells highly sensitive to cytotoxicity induced by VP-16. Lentiviral infections and cytotoxic treatments were as described for panel B. VP-16-treated (5 µM) and untreated (UT) cells are shown. Forty-eight hours after the addition of VP-16 to the culture medium, cell viability was assessed by microscopic inspection, using an Axiovert S-100 Zeiss microscope with a 10x objective. (D) ELISAs showing cell death in VP-16-treated and untreated PC-3 cells expressing either Twist-1-specific or Mut-3 shRNA, as indicated. Cells were left untreated (–) or treated with the indicated concentrations of VP-16 for 48 h, and ELISAs were preformed as described in the legend to Fig. 2B. Cell death is expressed as arbitrary units, and values are means ± standard deviations from three independent experiments. (E) Percentages of survival of PC-3 cells infected with pLL lentivirues expressing Twist-1-specific or control (Mut-3) shRNA as described for panel B and then left untreated or treated with cisplatin (100 µM) for 22 h as indicated. Viability was extrapolated from the cultures used to obtain the results depicted in panel F, as for panel B, by normalizing numbers of live cells in the cisplatin-treated cultures to the numbers of live cells in the corresponding untreated cultures. Values are the means ± standard deviations of three independent measurements. (F) Light microscopic images showing that Twist-1 shRNA markedly sensitizes PC-3 cells to cisplatin-induced cytotoxicity. Cells were infected with pLL lentivirues and then left untreated (UT) or treated with cisplatin (100 µM) as shown. After a 22-hour treatment, cell viability was assessed by microscopic inspection, using an Axiovert S-100 Zeiss microscope with a 10x objective. (G) PI nuclear staining of PC-3 cells expressing Twist-1-specific or control shRNAs after treatment with cisplatin (300 µM) or culture medium (–) for 17 h. The assays show that cisplatin-induced DNA fragmentation in PC-3 cells is markedly enhanced by the knockdown of Twist-1. Lentiviral infections and cell plating were as described for panel E. PI nuclear-staining assays were performed as described for Fig. 2C, and DNA content was determined by using FCM followed by analysis with the FlowJo software. Percent survival reflects the fractions of live cells (i.e., containing at least G1 DNA) in untreated (–) and cisplatin-treated (300 µM) cultures, as indicated. Values are the means ± standard deviations of three independent measurements. (H) ELISAs showing cell death in cisplatin-treated and untreated PC-3 cells expressing either Twist-1-specific or Mut-3 shRNA, as indicated. Cells were left untreated (–) or treated with the indicated concentrations of cisplatin for 22 h, and ELISAs were preformed as described for Fig. 2B. Cell death is expressed as arbitrary units, and values are means ± standard deviations from three independent experiments.

To assess the relevance of Twist-1 to NF-B-dependent chemoresistance in cancer, we generated PC-3 lines expressing RelA-specific or control shRNAs. shRNA specificity and RelA silencing were verified as described before by Western blotting (Fig. 7A and data not shown [36]). Upon treatment with daunorubicin, Twist-1 mRNAs were markedly upregulated in control cells, as expected (Fig. 7B, Mut-3 lanes). Remarkably, however, this upregulation was virtually abolished by the silencing of RelA, indicating that it depended on NF-B-RelA dimers (Fig. 7B). RelA knockdown also slightly lowered basal Twist-1 levels (Fig. 7B). We concluded that the upregulation of Twist-1 by anticancer drugs in certain tumor cells is controlled by NF-B and that this upregulation in these cells is essential to counter cytotoxicity induced by these drugs. Hence, Twist-1 appears to be a key participant in NF-B-mediated chemoresistance in certain cancers.

View larger version (48K): [in this window] [in a new window]   FIG. 7. Upregulation of Twist-1 by daunorubicin requires NF-B. (A) Western blots showing the downregulation of RelA proteins in PC-3 cells infected with pLL lentivirues expressing RelA-specific, but not control (lane Mut-3), shRNA. ß-Actin is shown as a loading control. (B) RT-PCR showing kinetics of upregulation of Twist-1 mRNAs following treatment with daunorubicin (3 µM) in PC-3 cells expressing control shRNAs (lanes Mut-3) but not in cells expressing RelA-specific shRNAs. Times (in hours) reflect the duration of treatment with daunorubicin prior to extraction of the total RNA. Also shown, as a loading control, are the ethidium bromide-stained ß-actin PCR products.

Twist factors mediate a novel protective mechanism that is activated by NF-B.

View larger version (40K): [in this window] [in a new window]   FIG. 8. Twist-1 and Twist-2 fail to modulate the activation of the JNK pathway by TNF- or daunorubicin in RelA–/– cells. (A) JNK kinase assays (top) showing kinetics of JNK induction in control-, Twist-1-expressing-, and Twist-2-expressing RelA–/– cells (MIGR1, MIGR1-Twist-1, and MIGR1-Twist-2 lanes, respectively) after exposure to TNF- (100 U/ml) plus CHX (0.1 µg/ml) for the times indicated. Extracts were prepared from treated and untreated cells and then analyzed for JNK activity as detailed in reference 33. Briefly, JNK was immunoprecipitated with an anti-JNK-specific antibody and then subjected to kinase assays using GST-c-Jun as the substrate in the presence of [-32P]ATP (top). Western blots showing total JNK levels in the extracts as the loading controls (bottom). (B) JNK kinase assays (top) showing kinetics of JNK induction in control, Twist-1-expressing-, and Twist-2-expressing RelA–/– cells (MIGR, MIGR1-Twist-1, and MIGR1-Twist-2 lanes, respectively) after exposure to daunorubicin (5 µM) for the times indicated. Extracts were prepared from treated and untreated cells and analyzed for JNK activity as described for panel A. Western blots showing total JNK levels as the loading control (bottom).

To clarify this issue, we investigated the p53 status of our immortalized RelA^–/– fibroblast lines. As shown in Fig. 9A, p53 polypeptides were present in these cells at constitutively high levels—a sign of defective p53 function (28). Further, p53 expression was unchanged following exposure to daunorubicin (Fig. 9A), which normally stabilizes wild-type p53 polypeptides, thereby increasing their levels (28). Upon treatment with this agent, RelA null cells also failed to upregulate the p53 targets p21, Bax, and MDM2 (28, 53), as shown by Western blotting (Fig. 9A). These data are consistent with previous findings that immortalized (i.e., 3T3) fibroblasts are often deficient in p53 function (11, 15, 24). As expected, the expression of Twist-1 had no significant effect on the levels of either p53 or its downstream targets (Fig. 9A). Hence, in our immortalized RelA^–/– 3T3 lines, the p53 tumor suppressor activity is functionally compromised and seemingly unaffected by Twist proteins.

View larger version (73K): [in this window] [in a new window]   FIG. 9. The protective activity of Twist factors against daunorubicin-induced killing is independent of an interference with the p53 and p19ARF pathways and involves instead a suppression of inhibitory Bcl-2 phosphorylation. (A) p53 function is compromised in immortalized RelA–/– 3T3 fibroblasts. RelA–/– 3T3 fibroblasts fail to upregulate p53 and p53 targets genes following exposure to daunorubicin. Western blots showing levels of p53 and the p53 targets p21, MDM2, and Bax in MIGR1- and MIGR1-Twist-1-transduced RelA–/– fibroblasts prior to and after exposure to the indicated doses of daunorubicin. At the times shown, cell extracts were prepared and analyzed by immunoblotting. Levels of none of the examined polypeptides were upregulated by these treatments. The specific antibodies used are labeled on the left-hand side. p21 levels were almost undetectable even after exposure to daunorubicin, despite the fact that these levels were readily detected in control extracts (data not shown). ß-Actin is shown as a loading control. (B) Light microscopic images showing that the expression of Twist-1 and Twist-2 effectively rescues p19ARF–/– cells from daunorubicin-induced cytotoxicity. p19ARF–/– cells were infected with the indicated retrovirues, seeded onto 60-mm dishes as described for Fig. 2A, and then left untreated (UT) or treated with daunorubicin (5 µM) as shown. After a 15-hour treatment, cell viability was assessed by microscopic inspection, as detailed in the legend to Fig. 4B, using an Axiovert S-100 Zeiss microscope with a 10x objective. (C) Percentages of survival calculated for the cultures depicted in panel B. The viability of these cells was extrapolated as described for Fig. 4C by normalizing numbers of live p19ARF–/– cells in the daunorubicin-treated cultures to the numbers of live cells in the corresponding untreated cultures. Values are the means ± standard deviations of three independent measurements. (D) Comparable infection efficiencies of the p19ARF–/– cells used to obtain the results shown in panels B and C. FCM histogram profiles showing eGFP positivity in the MIGR1-, MIGR1-Twist-1-, MIGR1-Twist-2-infected p19ARF–/– cells used to obtain the results shown in panels B and C and uninfected p19ARF–/– controls (No Infection), as indicated. Cells were harvested after infection as described for Fig. 5D, and eGFP positivity was assessed by performing FCM followed by analysis with the FlowJo software (detailed in the legend to Fig. 2B). The bar indicates fractions of GFP+ cells. (E) Western blots showing that Twist-1 expression prevents daunorubicin-induced Bcl-2 phosphorylation on Ser-87 in RelA–/– 3T3 cells. RelA–/– cells were transduced with either MIGR1 or MIGR1-Twist-1 retroviruses, as indicated, and left untreated or treated with daunorubicin (5 µM) for the times shown. Antibodies and intact and cleaved caspase-3 products are indicated on the left- and right-hand sides, respectively. ß-Actin is shown as the loading control. (F) Twist-1 is essential to prevent Bcl-2 phosphorylation induced by anticancer drugs. Western blots showing levels of total and phospho-Bcl-2 in VP-16-treated PC-3 cells expressing either Twist-1-specific or Mut-3 shRNAs, as indicated. Transduced PC-3 lines were the same as those used to obtain the results shown in Fig. 6B to D and were left untreated (0-hour time points) or treated with VP-16 (5 µM) for the times indicated. The antibodies used are labeled on the left-hand side. ß-Actin is shown as the loading control. (G) Western blots showing levels of total and phosphorylated Bcl-2 (on Ser-87) in MIGR1- and MIGR1-Twist-1-infected RelA null cells that were either left untreated (0-h time points) or treated with TNF- and CHX as described for Fig. 3A. MIGR1- and MIGR1-Twist-1-transduced cells were as indicated. The antibodies used are labeled on the left-hand side. ß-Actin is shown as the loading control. n.s., nonspecific.

Finally, because of their ability to block both the extrinsic and intrinsic pathways of PCD, we investigated whether the Twist-1 factors had any effect on the expression and/or function of Bcl-2-family proteins. In MIGR1-transduced cells, Bcl-2 levels were modestly upregulated by daunorubicin treatment, and this upregulation was accompanied by an apparent upward shift of the Bcl-2-specific band. Surprisingly, however, neither this upward shift nor this modest upregulation was observed in MIGR1-Twist-1-transduced cells (Fig. 9E). To verify whether these daunorubicin-induced changes of Bcl-2 proteins in MIGR1-infected cells were owed to phosphorylation, we preformed Western blotting using a phospho-specific antibody capable of detecting Bcl-2 phosphorylation on Ser-87, which inhibits the protective activity of Bcl-2 (1, 39). Remarkably, the ectopic expression of Twist-1 markedly suppressed the daunorubicin-elicited phosphorylation of Bcl-2 on Ser-87, whereas this phosphorylation of Bcl-2 was readily detected in control cells (Fig. 9E, MIGR1 lanes). These effects of Twist-1 on Bcl-2 correlated with an inhibition of caspase-3 activation (a key event in apoptosis signaling), as shown by a reduced appearance of p12/p17 cleavage products in MIGR1-Twist-1-infected cells (Fig. 9E). Interestingly, Bcl-2 proteins also block necrosis signaling (19), and so these effects of Twist-1 on Bcl-2 phosphorylation may also account for the suppression of this signaling. Twist-1 had no apparent effect instead on the proapoptotic member of the Bcl-2 family, Bad (Fig. 9E). These findings provide a basis for the protective activity of Twist-1 against chemotherapy-induced PCD.

Indeed, the physiological relevance of this inhibitory activity of Twist-1 on Bcl-2 phosphorylation is underscored by findings for knockdown systems. Whereas Twist-1 silencing had no effect on basal Bcl-2 phosphorylation on Ser-87 or on the modest dephosphorylation seen in PC-3 cells shortly after treatment with VP-16 (Fig. 9F, lanes for 4.5 and 9 h), this silencing markedly enhanced Bcl-2 phosphorylation at later times (Fig. 9F, lane for 18 h; compare the results for Twist-1 and Mut-3 shRNAs). Conversely, Twist-1 knockdown had no apparent effect on total Bcl-2 levels (Fig. 9F). Hence, Twist-1 plays an essential role in certain cancer cells in preventing inhibitory Bcl-2 phosphorylation induced by chemotherapeutic drugs. Thus, we investigated whether these effects of Twist-1 on Bcl-2 could also account for Twist-1-afforded suppression of TNF--induced PCD. In MIGR1-transduced RelA null cells, Bcl-2 was effectively phosphorylated following treatment with TNF-, and this phosphorylation was virtually abrogated by the expression of Twist-1 (Fig. 9G, MIGR1-Twist-1 lanes). In this system, Twist-1 expression did not affect total Bcl-2 levels, with modest effects being observed only at late times (Fig. 9G). Hence, Twist-1 is required to prevent chemotherapy (and TNF-)-induced Bcl-2 phosphorylation on Ser-87, and this inhibitory activity may account for its marked protective effects against both necrotic and apoptotic PCD.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Here, we have identified Twist-1 as a new mediator of the protective activity of NF-B. Twist-1 i