MEKK1-Induced Apoptosis Requires TRAIL Death Receptor Activation and Is Inhibited by AKT/PKB Through Inhibition of MEKK1 Cleavage

MEKK1-induced apoptosis requires TRAIL death receptor activation and is inhibited by AKT/PKB through inhibition of MEKK1 cleavage

Andrea H Bild1, Francisco J Mendoza3, Erika M Gibson3, Mei Huang2, Jacylyn Villanueva3, Timothy P Garrington1, Richard Jove2, Gary L Johnson1 and Spencer B Gibson*,3

1Department of Pharmacology, University of Colorado, 2400 East Ninth Street, Denver, Colorado, CO 80262, USA; 2Molecular Oncology Program, H Lee Moﬃtt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida, FL 33612, USA; 3Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0V9

MEK kinase 1 (MEKK1) induces apoptosis through the participates in regulation of the c-Jun N-terminal activation of caspases. The mechanism for MEKK1- kinase (JNK) and extracellular signal regulated kinase induced apoptosis involves caspase-mediated cleavage of (ERK) pathways (Lange-Carter et al., 1993; Widmann MEKK1, releasing a pro-apoptotic 91 kDa kinase frag- et al., 1999). MEKK1 is also involved in induction of ment that serves to further amplify caspase activation in a apoptosis through the activation of caspases feedback loop. Both cleavage of MEKK1 and increased (Widmann et al., 1997, 1999). Exposure of cells to expression of death receptor 4 (DR4, TRAILR1) and stresses, such as genotoxins, activates caspase 3-like death receptor 5 (DR5, TRAILR2) occur following proteases, which cleave MEKK1 into a pro-apoptotic exposure of cells to genotoxins. Overexpression of kinase 91 kDa kinase domain fragment (Widmann et al., inactive MEKK1 inhibits MEKK1-mediated apoptosis and 1998). This cleavage of MEKK1 releases its 91 kDa effectively blocks death receptor upregulation following form from the membrane fractions into the cytoplasm etoposide treatment. Herein, we investigate the role of (Deak et al., 1998; Schlesinger et al., 1998). The death receptor activation and the ability of AKT/PKB 91 kDa kinase fragment further amplifies caspase (AKT) to inhibit cell death in MEKK1-induced apoptosis. activation in a feedback loop and is a strong inducer We show that by preventing DR4 and DR5 activation of apoptosis (Cardone et al., 1997; Widmann et al., through expression of decoy receptor 1 (DcR1) and 1998). Inhibitors of caspases block this cleavage, and dominant negative FADD, we inhibit MEKK1-induced mutation of the consensus caspase 3-like cleavage site apoptosis. Furthermore, expression of 91 kDa MEKK1 of MEKK1 inhibits both its cleavage and its ability to increased DR4 and FAS mRNA and protein levels. induce apoptosis. Both cleavage of MEKK1 and MEKK1-induced apoptosis is amplified by blocking PI-3 increased expression of DR4 and DR5 occur follow- kinase activation and overexpression of AKT blocked both ing exposure of cells to genotoxins (Gibson et al., MEKK1-induced apoptosis and caspase activation. AKT 2000). overexpression also prevented the cleavage of endogenous Overexpression of a kinase inactive MEKK1 inhibits MEKK1 by genotoxins. AKT did not, however, block MEKK1-mediated apoptosis and effectively blocks MEKK1-induced JNK activation, showing that regulation death receptor upregulation following etoposide treat- of the JNK pathway by MEKK1 is independent of its role ment (Gibson et al., 2000). Thus, cleavage of MEKK1 in regulation of apoptosis. Thus, MEKK1-induced serves to activate a cell death-promoting response. apoptosis requires TRAIL death receptor activation and MEKK1 is emerging as an important mediator of is blocked by AKT through inhibition of MEKK1 cleavage. apoptosis in human cancers. MEKK1 is necessary for Oncogene (2002) 21, 6649 – 6656. doi:10.1038/sj.onc. the induction of apoptosis following anoikis and 1205819 specific genotoxic treatments (Cardone et al., 1997; Gibson et al., 1999). MEKK1 is also involved in Keywords: apoptosis; serine threonine kinases; signal apoptosis of colon cancer cells following chemotherapy transduction; death receptors; caspases; MAPK (Soh et al., 2001). Furthermore, aberrant MEKK1 cleavage and subsequent apoptosis in certain ovarian adenocarcinomas leads to drug resistance (Gebauer et Introduction al., 2000). Dysregulation of apoptosis can also occur by mutation of genes involved in the death receptor MEK kinase 1 (MEKK1) is a 196-kDa mitogen- pathways. Death receptors such as DR4, DR5, Fas, activated protein kinase (MAPK) kinase kinase that and tumor necrosis factor (TNF) receptor belong to the superfamily of TNF receptors that initiate apoptotic signals upon ligation. Activation of these *Correspondence: SB Gibson; E-mail: [email protected] death receptors leads to recruitment of other proteins, Received 26 November 2001; revised 19 June 2002; accepted 28 including FADD (an adaptor protein) and caspase 8. June 2002 Association of adaptor proteins with the death receptor AKT blocks MEKK1 induced apoptosis AH Bild et al 6650 leads to caspase 8 activation, which in turn leads to the Results activation of caspase 3-like molecules and eventual apoptotic cell death (Ashkenazi and Dixit, 1999; Inhibition of death receptor activation prevents Golstein, 1997; Green, 2000). Inactivating mutations MEKK1-induced apoptosis in DR4 and DR5 have been characterized in certain metastatic breast cancer and non-Hodgkin’s lympho- Decoy receptors compete with specific death receptors mas (Lee et al., 2001; Shin et al., 2001). Further, Fas for ligand binding (Ashkenazi and Dixit, 1999). mutations occur in specific malignant lymphomas and Overexpression of DcR1 blocks ligand binding and solid tumors (Mullauer et al., 2001; Straus et al., 2001). activation of DR4 and DR5 (Pan et al., 1997; Sheridan Other TNF receptor family members, such as decoy et al., 1997). To determine if DR4 and DR5 activation receptor 1 (DcR1), bind to ligands for death receptors is involved in MEKK1-induced apoptosis, human but, lacking the cytoplasmic domains to recruit pro- embryonic kidney (HEK) 293 cells overexpressing apoptotic proteins, fail to induce apoptosis (Sheridan MEKK1 were transfected with vector alone or with et al., 1997). By competitive inhibition, these decoy DcR1 cDNA. The extent of apoptosis was determined receptors act to circumvent death receptor activation, by TdT staining in a Tunel assay, as described in the thereby negatively regulating death receptor-induced Materials and methods section. HEK-293 cells contain- signaling and apoptosis. ing empty vector showed 21% apoptosis when Another protein that negatively regulates apoptosis MEKK1 was expressed, compared with 4% apoptosis is the AKT/PKB (AKT) proto-oncogene. AKT is in cells expressing DcR1 in the presence of MEKK1 found upregulated in many cancers, including breast, (Figure 1a). HEK-293 cells were also transiently ovarian, prostate, and pancreatic malignancies (Blume- transfected with MEKK1 in the absence or presence Jensen and Hunter, 2001). AKT mediates cell survival of FADD DN, which acts to block caspase 8 by suppressing apoptosis induced by a variety of activation following ligand binding of death receptors, apoptotic stimuli, including loss of cell adhesion, including DR4 and DR5. Expression of MEKK1 alone growth factor withdrawal, and exposure to genotoxins. caused 43% apoptosis. In the presence of FADD DN, The action of AKT-mediated protection occurs both however, MEKK1 induced only 20% apoptosis, a by inhibition of pro-apoptotic proteins, such as caspase 9, BAD, and the Forkhead transcription factor, and by activation of anti-apoptotic proteins, such as the NF-kB and CREB. AKT can also promote cell survival through inactivation of caspase-mediated apoptotic signaling (Datta et al., 1999; Khwaja, 1999). Activation of AKT has been shown to block caspase activation and apoptosis following treatment with many different apoptotic stimuli, including anoikis and genotoxic agents, and therefore potentially contributes to chemotherapy resistance in some forms of cancer. Recent studies suggest a role of AKT in inhibitory modulation of TNF-receptor mediated apoptosis. AKT is also implicated in Fas-mediated apoptosis as Pten +/7 mice show decreased Fas- induced apoptosis (Di Cristofano et al., 1999). Further, AKT regulates the expression of c-FLIP, a caspase 8 dominant negative that inhibits Fas death signals in certain tumor cells (Panka et al., 2001; Tschopp et al., 1998). To further define the mechanism of MEKK1- mediated apoptosis, we investigated the role death receptor pathway activation. Transfection studies involving overexpression of DcR1 and dominant negative FADD (FADD DN) show that death receptor signaling is required for MEKK1-mediated apoptosis. Inhibition of PI-3 kinase potentiates MEKK1-induced Figure 1 MEKK1-induced apoptosis in HEK-293 cells overex- apoptosis, and expression of AKT blocks initial pressing DcR1 and FADD DN. (a) HEK-293 lines stably expressing DcR1 or vector alone were transiently transfected with cleavage of endogenous MEKK1 to its pro-apoptotic MEKK1. Forty-eight hours following transfection, the cells were 91 kDa form and subsequent MEKK1 caspase activa- stained for MEKK1 and TdT. The percentage of TdT positive tion and apoptosis. Thus, MEKK1-induced apoptosis cells expressing MEKK1 determined the percent apoptosis. (b) requires death receptor activation and is blocked by HEK-293 cells were transiently transfected with MEKK1 in the presence or absence of FADD DN. Per cent apoptosis was deter- AKT through inhibition of caspase activation, which mined by acridine orange staining. Control cells were untrans- inhibits cleavage of MEKK1 to its pro-apoptotic fected or transfected with FADD DN alone. Results are 91 kDa form. representative of three independent experiments

Oncogene AKT blocks MEKK1 induced apoptosis AH Bild et al 6651 percentage similar to that seen in untransfected cells (Figure 1b). Cells expressing FADD DN without MEKK1 also showed no increase in apoptosis above control levels. These results suggest that MEKK1- induced apoptosis involves activation of death receptor signaling pathways.

MEKK1 upregulates DR4 and Fas mRNA and protein levels To determine if MEKK1 directly affects the mRNA expression levels of TNF death receptor pathway members, RNAse protection assays (RPAs) were performed. HEK-293 cells expressing the 91 kDa MEKK1 kinase domain were lysed and RNA was extracted. RPA analysis using the hApo3d panel was performed as described in the Materials and methods section. Cells transfected with 91 kDa MEKK1 show a 2.7-fold increase in Fas mRNA expression and a 1.6- fold increase in DR4 mRNA levels when compared to cells expressing a vector control (Figure 2a). Western blot analysis reveal that transfection of 91 kDa MEKK1 increased Fas protein levels by twofold and DR4 protein levels by fourfold when compared to cells transfected with vector alone (Figure 2b). Caspase 8 mRNA levels increased threefold following 91 kDa MEKK1 overexpression but failed to show significant increase in protein levels. No increase in DR5 mRNA Figure 2 MEKK1 upregulates DR4 and Fas mRNA and protein and protein levels following transfection of 91 kDa levels. HEK-293 cells were transfected with control vector or a 91 kDa MEKK1 vector. Thirty-six hours following transfection, MEKK1 in cells was detected (Figure 2b). Therefore, RPA analysis was carried out as described in Materials and meth- MEKK1 leads to upregulation of the DR4 and Fas ods. Fold increases in (a) DR4 and (b) Fas were quantitated and death receptors. standard deviation performed. (c) Western blotting was performed with HEK-293 cells transfected with 91 kDa MEKK1 or vector control. Twenty-four hours post-transfection, cells were Inhibition of PI-3 kinase potentiates MEKK1-induced lysed as described and blotted for DR4, DR5, Fas and caspase 8. apoptosis The level of expression was determined using a STORM phosphorimager using enhanced chemifluorescence (ECF). The expres- In order to further define the regulation of MEKK1- sion was equalized for loading variations by re-probing with b- induced apoptosis, PI-3 kinase activity was inhibited, actin and the ability of MEKK1 to promote apoptosis was examined. PI-3 kinase is important in cellular survival and has been found to activate AKT and mediate protection of cell from apoptosis. HEK-293 cells were treated with 200 nM of a PI-3 kinase inhibitor, wortmannin, for 12 h, and apoptosis was determined as described above. Expression of MEKK1 alone induced apoptosis in 39% of cells transfected. MEKK1 expression in the presence of wortmannin induced apoptosis in 52% of cells (Figure 3). Thus, by inhibition of endogenous PI-3 kinase, apoptosis induced by overexpression of MEKK1 was potentiated by 24%. This result suggests that even basal levels of PI-3 kinase activity can protect cells by negative regulation of MEKK1-induced apoptosis.

AKT blocks MEKK1-induced apoptosis and caspase Figure 3 Inhibition of PI-3 kinase potentiates MEKK1-induced 3-like protease activation apoptosis. HEK-293 cells were transfected with a MEKK1-green AKT is activated by PI-3 kinase and blocks genotoxin- fluorescent protein (GFP) vector or control GFP vector. Twenty- four hours after transfection, cells were serum starved for 12 h induced apoptosis (Khwaja, 1999). Since MEKK1 is with DMSO or 200 nm Wortmannin. Cells were then fixed using involved in genotoxin induced apoptosis, the ability of 3.8% paraformaldehyde and quantified in a blinded manner for AKT to block MEKK1-induced apoptosis was exam- expression of GFP protein and apoptotic nuclei

Oncogene AKT blocks MEKK1 induced apoptosis AH Bild et al 6652 ined. HEK-293 cells were transiently transfected with full length MEKK1 in the presence or absence of a constitutively activated myristoylated AKT (myr- AKT), wild-type AKT (wt-AKT), p35, or vector alone and stained for protein expression using anti-MEKK1 and anti-AKT antibodies. p35 is a protein previously shown to inhibit MEKK1 cleavage and apoptosis and thus acts as a control in these experiments. Expression of myr-AKT and wt-AKT resulted in increased AKT kinase activity as determined by an in vitro AKT kinase assay (data not shown). Per cent apoptosis was quantified using a TdT-based TUNEL assay. The number of cells expressing MEKK1 and staining positively for TdT were then counted by fluorescence microscopy. At least 400 cells were counted for each condition in three separate experiments. Forty-two per cent of cells expressing MEKK1 with pCMV5 empty vector were apoptotic. In cells co-expressing MEKK1 with either myr-AKT or wt-AKT, however, the percentage of apoptotic cells was 8.5% and 7.6%, respectively (Figure 4a). These results reflect an 80% inhibition of MEKK1-induced apoptosis by AKT. As an internal control, cells co-expressing p35, an inhibitor of caspases, had 14.7% apoptosis (Figure 4a). Thus, AKT strongly inhibits MEKK1-induced apoptosis. AKT blocks apoptosis by multiple mechanisms, including activation anti-apoptotic proteins and by inhibition of pro-apoptotic proteins. To determine if AKT specifically blocks MEKK1-induced caspase activation, HEK-293 cells were transiently transfected with full length MEKK1, with or without the expression of myr-AKT and wt-AKT. Caspase activity was determined using a caspase 3 consensus substrate (DEVE-AFC), which, when cleaved, produces a fluorescent product quantified as described, in the Materials and methods section. Expression of MEKK1 alone caused a 2.1-fold increase in caspase 3-like protease activity above baseline. This fold increase Figure 4 Expression of myr-AKT and wt-AKT inhibit MEKK1- was reduced to below basal levels by co-expression induced apoptosis and caspase activation. (a) Cells were analysed with myr-AKT (0.87-fold) and to basal level by co- by fluorescence microscopy for expression of MEKK1 and for po- expression with wt-AKT (1.07-fold) (Figure 4b). These sitive TdT staining. The percentage of cells with apoptotic nuclei as determined by TdT staining for each condition was quantified results are consistent with the hypothesis that AKT in a blinded manner. At least 400 cells were counted for each con- blocks MEKK1-induced apoptosis by inhibiting dition in three separate experiments. (b) HEK-293 cells expressing caspase 3-like protease activation. full-length MEKK1 along with myr-AKT, wt-AKT, or p35 were lysed. Caspase activity was determined by measurement of pro- duction of a fluorescent cleavage product of a caspase 3 consensus AKT inhibits cleavage of endogenous MEKK1, which substrate (DEVE-AFC), as described in the Materials and meth- requires caspase 3 proteases ods section. Levels of caspase activation are expressed as fold dif- ference in comparison with cells expressing pCMV5 vector alone Endogenous MEKK1 is cleaved by caspase 3-like proteases following treatment with genotoxic agents. HEK-293 cells stably expressing myr-AKT or vector cells expressing pCMV5 or b-gal alone (Figure 5b). alone were treated with etoposide (100 mM)orUV-C These results show that AKT overexpression blocks (40 J/m2). Following treatment, cells were lysed and initial cleavage of endogenous MEKK1 to its assayed for full-length endogenous MEKK1 by pro-apoptotic 91 kDa kinase domain fragment. To Western blotting. Forty-eight hours following etopo- further define the specific caspase involved in MEKK1 side treatment, cleavage of endogenous MEKK1 was cleavage, we investigated primary mouse embryonic inhibited in the presence of a constitutively active myr- fibroblasts (MEFs) with homozygous deletion of AKT when compared to cells expressing vector alone caspase 3. MEF caspase 37/7 cells were treated with (Figure 5a). Following UV irradiation, inhibition of etoposide for 24 and 48 h, and Western blotting was cleavage of full-length MEKK1 was also seen in cells performed to detect the presence of uncleaved, expressing myr-AKT and wt-AKT as compared with endogenous full-length MEKK1. As shown in Figure

Oncogene AKT blocks MEKK1 induced apoptosis AH Bild et al 6653 induced apoptosis by blocking activation of the JNK pathway, HEK-293 cells were transiently transfected with MEKK1 in parental cells or in cells stably expressing myr-AKT. The level of JNK activation was determined as described in the Materials and methods section. Transfection of MEKK1 into parental cells or cells expressing myr-AKT led to equivalent levels of JNK activation (Figure 6). Myr-AKT or vector alone did not independently activate JNK activity (Figure 6, data not shown). Thus, AKT does not block MEKK1-induced JNK activation.

Discussion

Our results indicate that death receptor activation is required for MEKK1-mediated apoptosis. Cleavage of MEKK1 by caspase-3 proteases precedes MEKK1- induced death in response to chemotherapy treatment. The mechanism by which MEKK1 initiates apoptosis involves upregulation of DR4 and Fas death receptor levels. Expression of kinase inactive MEKK1 blocks the upregulation of DR4 following etoposide treatment (Gibson et al., 2000). MEKK1 activation also increases Fas ligand expression in Jurkat T cells, suggesting that MEKK1 regulation of death receptor activation is an important mechanism for induction of apoptosis in diﬀerent cell types (Faris et al., 1998a,b). Our results

Figure 5 Cleavage of endogenous MEKK1 following etoposide or ultraviolet radiation in HEK-293 cells expressing AKT. HEK-293 cells were treated with 100 mM etoposide or ultraviolet irradiation (UV). Forty-eight hours later, the cells were lysed as described in Materials and methods section, and Western blots for endogenous MEKK1 were performed. (a) HEK-293 cells stably expressing empty vector or myr-AKT were treated with etoposide for 48 h and Western blots for MEKK1 were performed. The blots were stripped and reprobed with b-actin antibodies. (b) HEK-293 cells were transiently transfected with vector alone, wt-AKT, or myr-AKT and treated with 40 J/m2 UV irradiation. The cells were then lysed, and Western blotting for endogenous MEKK1 was performed. (c) Caspase 3 7/7 MEFs were treated with 100 mM etoposide or DMSO alone for 24 and 48 h, and Wes- tern blotting was performed for endogenous MEKK1 and b-actin

5c, cells lacking caspase 3 show no cleavage of full- length MEKK1, while MEKK1 in wild-type control cells undergoes protease cleavage. Thus, cleavage of MEKK1 into its pro-apoptotic 91 kDa domain speciﬁcally requires caspase 3 proteases. Figure 6 JNK activation following expression of MEKK1 in Prevention of MEKK1-induced apoptosis by AKT is not HEK-293 cells. Parental or cells stably expressing myr-AKT were mediated by inhibition of JNK activation transfected with MEKK1 cDNA of empty vector and analysed for JNK activity in a GST-c-Jun assay. (a) Phosphorylated MEKK1 is known to active c-Jun N-terminal kinase GST-c-Jun following JNK kinase assay as described in Materials and methods section was detected by phosphorimager. (b) Kinase (JNK), a MAP kinase family member. JNK activation activity was quantiﬁed by phosphorimaging analysis of the extent has been postulated to be involved with the induction of phosphorylation of GST-c-Jun. Standard error was calculated of apoptosis. To determine if AKT blocks MEKK1- from three independent experiments

Oncogene AKT blocks MEKK1 induced apoptosis AH Bild et al 6654 indicate that MEKK1 activates death receptor apopto- Activation of MEKK1 leads to JNK activation. tic signaling pathways to induce apoptosis. JNK has been implicated in the induction of apoptosis. Inhibition of an upstream regulator of AKT In the presence of AKT, MEKK1 mediated JNK activation increases MEKK1-induced apoptosis. AKT activation was not affected. This result suggests that blocks MEKK1-induced apoptosis caspase amplifica- JNK activation is not responsible for MEKK1’s pro- tion by inhibiting cleavage of MEKK1 by caspase 3, apoptotic effects and that AKT’s prevention of thereby preventing release of the pro-apoptotic 91 kDa MEKK1-induced apoptosis is unrelated to MEKK1’s kinase fragment of MEKK1. This provides evidence regulation of JNK. It is still possible, however, that that the apoptotic effects of MEKK1 are mediated by AKT could block downstream events following JNK its cleavage into a 91 kDa kinase fragment. Corre- activation leading to inhibition of apoptosis. Since spondingly, it has been shown that caspase-3 is expression of kinase inactive MEKK1 fails to block required for programmed cell death in fibroblasts in JNK activation by etoposide (data not presented) but response to chemotherapy treatment and for apoptosis effectively blocks etoposide-induced apoptosis, it is in ES cells following UV irradiation (Woo et al., unlikely that JNK is playing a major role in MEKK1- 1998). induced apoptosis. Indeed, MEKK1 knock out fibro- The direct mechanism by which AKT blocks blasts show a decreased JNK response to cell stresses MEKK1 induced caspase activation is unknown, but that alter the cytoskeletal structure and an increased our findings indicate that it is by inhibiting caspase 3 apoptotic response to these stresses, suggesting that activity and thereby blocking subsequent MEKK1 MEKK1-induced JNK activation may actually be cleavage to its 91 kDa kinase fragment. One mechan- protective (Yujiri et al., 1998). ism by which caspase 3 activity is blocked is by the It is becoming more important to characterize the inhibitor of apoptosis (IAP) family of anti-apoptotic relationship between apoptotic and oncogenic signaling proteins. Cisplatin causes activation of caspase 3 and pathways in order to tailor more targeted therapies for caspase 9 and is blocked by XIAP overexpression cancer cells. Knowledge of the mechanisms by which (Asselin et al., 2001). Further, the IAP family members cells regulate apoptosis can lead to potentiation of XIAP and survivin are induced by VEGF in these pathways using specialized chemotherapies. endothelial cells. VEGF is important in cell survival Further, delineation of oncogenic pathways that during angiogenesis and vasculogenesis in part due to subvert apoptotic pathways or effects of chemother- AKT activation (Tran et al., 1999). AKT is also apeutic reagents will allow generation of more targeted implicated in NF-kB modulation of XIAP levels, cancer treatments to block these effects. conferring resistance to apoptosis in macrophages Cumulatively, our results show that death receptor (Lin et al., 2001). Thus, AKT may affect activation activation plays an important role in MEKK1-induced of proteins involved in caspase 3 inhibition, thereby apoptosis and that AKT blocks initial MEKK1 blocking MEKK1’s ability to generate apoptotic cleavage and subsequent induction of caspase activa- signaling by upregulation of death receptors. tion and apoptosis. Inhibiting the upstream activator Evidence suggests that death receptor activation of AKT, PI-3 kinase, potentiates MEKK1-induced plays a role in genotoxin-induced apoptosis. For apoptosis. Our findings identify potential molecular example, increased expression of Fas ligand following targets that may be effective at regulating TNF doxorubicin treatment contributes to the induction of receptor and MEKK1-mediated apoptosis and over- apoptosis (Friesen et al., 1996). Treatment with coming survival signals such as AKT, leading to more etoposide results in increased expression of DR4 and targeted cancer therapies. DR5 in both breast and lung cancer cells and is a proposed mechanism for genotoxin-induced apoptosis (Gibson et al., 2000). Alternatively, inhibition of TRAIL binding to DR4 and DR5 reduces the Materials and methods apoptotic response to etoposide (Gibson et al., 2000). Further, preliminary studies have shown the ability of Reagents TRAIL to reduce the size of human tumors in mice. In Dr Thomas Franke generously contributed AKT constructs. combination with genotoxic agents, TRAIL can Constitutively active myristoylated c-AKT (myr-AKT) and eradicate some human tumors in mice and can lead wild type c-AKT (wt-AKT) were expressed in pCMV5. The to a synergistic apoptotic response in some breast MEKK1 constructs used have been previously described. cancer cell lines (Gibson et al., 2000; Griffith and HEK-293 cells were used for all transfections and for Lynch, 1998; Gura, 1997). These responses could at generation of AKT stable cell lines. least partially be explained by the upregulation of DR4 and DR5 expression. We have shown that AKT is AKT kinase assay effective at inhibition of MEKK1 and etoposide- HA-tagged myr-AKT was transfected by lipofectamine into induced apoptosis. Thus, blocking AKT or its HEK-293 cells and immunoprecipitated with anti-HA upstream regulators could potentially result in antibodies following serum starvation (0.1% BCS) for 12 h. enhancement of the apoptotic signal by TNF death The kinase assay was performed using a GSK-3 peptide receptor pathways, particularly cells with increased substrate in the presence of g32P-ATP and quantified by expression of AKT. scintillation counting.

Oncogene AKT blocks MEKK1 induced apoptosis AH Bild et al 6655 were transfected with 91 kDa MEKK1 using Superfect Apoptosis assay reagent (Qiagen) and lysed as described above. Hundred mg HEK-293 cells grown on glass coverslips were transfected of protein was loaded on a SDS page gel and Western blotted with appropriate constructs. Following 0.1% serum starva- with antibodies against DR4 (Santa Cruz), DR5 (Santa tion for 12 or 36 h, cells were fixed in 2% paraformaldehyde Cruz), caspase 8 (Upstate Biotech.), and Fas (Santa Cruz). and permeabilized with 0.2% Triton X-100. After washing in The blots were visualized using enhance chemiluminescence PBS, cells were blocked with filtered cultured medium and PLUS kit (Pharmacia) and analysed on a STORM incubated in TdT reaction mix (Boehringer). After washing, phosphorimager (Molecular Dynamics) for fluorescence. coverslips were incubated with the appropriate primary The blots were stripped and re-probed with anti-b-actin antibodies (rabbit anti-MEKK1, goat anti-AKT and Hoechst (Sigma) to determine equal loading and analysed as described stain). The coverslips were then washed and incubated with above. secondary antibodies (Cy5 conjugated goat anti-rabbit, FITC conjugated donkey anti-goat, and Cy3 conjugated streptavi- Caspase assay din). Images were collected using a Leica DMRXA microscope and analysed with SlideBook v2.0 software. Cells HEK-293 cells were co-transfected with 1.2 mg total DNA of were quantified in a blinded manner. full-length MEKK1 and myr-AKT, wt-AKT, p35, or pCMV5. Following serum starvation in 0.1% BCS, cells were lysed in 50 mM Tris (pH 7.4), 1 mM EDTA, and 10 mM RNAse protection assay digitonin for 10 min at 378C. Following a brief vortex, cell A RiboQuant Multi-Probe RNAse Protection Assay System lysates were centrifuged at 14 000 g for 10 min. Sixty mgof (Pharmingen) was used according to the manufacturer’s lysate proteins were incubated with 5 mM of a DEVE-caspase instructions. A hAPO3c probe set containing DNA templates substrate (Bachem). Generally, 0.5 mg lysates were used for for caspase 8, FASL, Fas, DR3, DcR1, DR4, DR5, TRAIL, Western blotting to confirm transfection levels. Fluorescence TNFR p55, TRADD, RIP, L32, and GAPDH (Pharmingen) was then monitored with an excitation wavelength of 380 nm was used for T7 RNA-polymerase direct synthesis of g32-P- and emission wavelength of 460 nm to determine activity by UTP-labeled anti-sense RNA probes. The probes were a luminometer. Fluorescence of the substrate alone was hybridized with 20 mg of RNA isolated from HEK-293 cells subtracted in each case. using RNAzol B (Tel-Test, Inc.). Samples were then digested with RNAse to remove single-stranded (non-hybridized) c-Jun kinase assay RNA. Remaining probes were resolved on denaturing polyacrylamide gels. Quantitation was made by phosphor- c-Jun kinase (JNK) activity was measured using a solid phase imager analysis. kinase assay in which glutathione-S-transferase-c-Jun (1-79) (GST-Jun) bound to glutathione-Sepharose 4B beads was used to affinity purify JNK from transfected cell lysates. Western blotting HEK-293 cells were lysed in 70 mM b-glycero-phosphate, HEK-293 cells transfected with myr-AKT, wt-AKT, and the 1mM EGTA, 100 mM Na3VO4,1mM DTT, 2 mM MgCl2, negative controls b-gal and pCMV5 were lysed in a TGH 0.5% Triton X-100, and 20 mg/ml of aprotinin. Following buffer (1% Triton X-100, 10% glycerol, 50 mM NaCl, 50 mM centrifugation at 10 000 g for 5 min, cleared lysates were HEPES, pH 7.3, 5 mM EDTA, 1 mM sodium orthovanadate, collected and protein concentration was normalized by the 1mM phenylmethylsulphonyl fluoride, 10 mg/ml leupeptine, Bradford assay. JNK phosphorylation was quantified using a and 10 mg/ml aprotinin) following UV irradiation (40 J/cm2) STORM phosphorimager. or etoposide treatment. After rotation at 48C for 30 min, lysates were centrifuged at 14 000 g for 30 min, supernatants were collected, and protein concentration was determined by Bradford analysis. Generally, 0.5 mg of protein was separated by 10% SDS – PAGE, and transferred to a Acknowledgements nitrocellulose membrane. Membranes were probed with AH Bild was supported by a DOD predoctoral fellowship MEKK1 specific antibodies generated in our lab or a C-22 DAMD17-98-1-8297. SB Gibson was supported in part by MEKK1 antibody (Santa-Cruz). In addition, HEK-293 cells Canadian Institutes of Health Research.

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