We previously reported that the anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 induces DiFi colon cancer cells to undergo apoptosis, and this apoptosis was accompanied by activation of the two apoptosis initiation caspases, caspase-8 and caspase-9. In the current study, we found that pretreatment of DiFi cells with the caspase-8-specific inhibitor z-IETD-fmk but not pretreatment with the caspase-9-specific inhibitor z-LEHD-fmk inhibited mAb 225-induced apoptosis, indicating that caspase-8 plays an essential role in initiating mAb 225-induced apoptosis. Because caspase-8 is activated primarily by the members of the tumor necrosis factor (TNF) receptor family, such as Fas, TNF receptor-1 (TNFR1), or receptors for TNF-related apoptosis-inducing ligand (TRAIL), we investigated whether mAb 225 activated caspase-8 by regulating one or more of these known pathways. Exposure of DiFi cells to TNFα or TRAIL activated caspase-8 and induced apoptosis in the cells. A TNFR1-antagonistic mAb or a TRAIL decoy receptor inhibited the activation of caspase-8 and the subsequent apoptosis induced by TNFα or TRAIL, respectively, in the cells. However, neither the TNFR1-antagonistic mAb nor the TRAIL decoy receptor inhibited mAb 225-induced activation of caspase-8 and apoptosis in DiFi cells. DiFi cells express detectable level of Fas but are not sensitive to the treatment by the Fas-agonistic mAb CH-11. A Fas-antagonistic mAb (ZB-4) inhibited the Fas-agonistic mAb CH-11-induced caspase-8 activation and apoptosis in Jurkat T-leukemic cells (used as positive control), but had no effect on mAb 225-induced activation of caspase-8 and apoptosis in DiFi cells. Taken together, our results suggest that mAb 225 does not interact with or regulate these known death receptor pathways. An exploration is therefore warranted for a novel mechanism by which mAb 225 activates caspase-8 and triggers apoptosis in DiFi cells.
Apoptosis, also known as programmed cell death, occurs in two phases, an initial commitment phase and a subsequent execution phase that manifests the typical apoptotic phenotype, which includes DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage and disassembly into membrane-enclosed vesicles (apoptotic bodies) (Nicholson and Thornberry, 1997; Thornberry et al., 1992). Caspases are a group of cysteinyl aspartate-specific proteinases that mediate highly specific proteolytic cleavage events in dying cells. Two major initiation pathways of caspase activation during apoptosis have been described (Ashkenazi and Dixit, 1998; Green and Reed, 1998; Sun et al., 1999; Thornberry and Lazebnik, 1998). The first pathway involves apoptosis mediated primarily by tumor necrosis factor (TNF) family death receptors (DR), such as TNF receptor-1 (TNFR1), Fas or the receptors for TNF-related apoptosis-inducing ligand (TRAIL), which contain a cytoplasmic death domain (DD). When Fas ligand (or TNFα) binds to Fas (or TNFR1), the adapter molecule FADD/Mort-1 is recruited to the receptors, allowing binding and autoactivation of procaspase-8 (Boldin et al., 1995, 1996; Chinnaiyan et al., 1995, 1996; Muzio et al., 1996, 1998; Yang et al., 1998). After caspase-8 is activated, it can process effector caspases (caspase-3, -6 or -7), inducing a cascade of caspase activation (Enari et al., 1996; Schlegel et al., 1996; Stennicke et al., 1998). In the second pathway, diverse proapoptotic signals converge at the mitochondrial level, provoking the translocation of cytochrome c from the mitochondria to the cytoplasm (Adachi et al., 1997; Bossy-Wetzel et al., 1998; Kharbanda et al., 1997; Kim et al., 1997; Kluck et al., 1997; Liu et al., 1996). Once cytochrome c (also known as Apaf-2) is in the cytoplasm, it binds to the apoptotic protease activating factor-1 (Apaf-1), which then permits recruitment of procaspase-9 (also known as Apaf-3) (Bossy-Wetzel and Green, 1999). The complex of cytochrome c, Apaf-1 and caspase-9 (called the ‘apoptosome’) is a critical activator of the effector caspases in the mitochondrial pathway.
We and others have shown previously that the anti-human epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 competes with EGF or transforming growth factor-α (TGFα) for receptor binding, prevents ligand-induced receptor tyrosine kinase activation, and inhibits the proliferation of a variety of cultured and xenografted human cancer cells that are stimulated by the TGFα/EGF receptor autocrine loop (Fan and Mendelsohn, 1998). In most of the cancer cell lines that we have studied, treatment with mAb 225 inhibits cell proliferation (Baselga et al., 1996; Fan et al., 1997; Peng et al., 1996; Stampfer et al., 1993; Van de Vijver et al., 1991). However, exposure of DiFi human colorectal cancer cells to mAb 225 results in cell death via apoptosis (Liu et al., 2000, 2001; Wu et al., 1995).
The anti-EGF receptor mAb-induced apoptosis in DiFi cells is characterized by activation of the initiation caspase-8 pathway, which was detected between 8 and 16 h after exposure of the cells to mAb 225 (Liu et al., 2000). The initiation caspase-9 was also activated, but this activation was not detected until a few hours after caspase-8 was activated. In the current study, we continued to investigate the involvement of these two initiation caspase pathways in mAb 225-induced apoptosis and found the activation of caspase-8 plays an essential role in initiating the apoptosis. Because caspase-8 is activated primarily by the TNF-family death receptor-mediated pathway, we examined whether mAb 225 activated caspase-8 by interacting or regulating one of these known pathways. We found that TNFα or TRAIL induced apoptosis in DiFi cells, and this apoptosis was inhibited by a TNFR1-antagonistic mAb or by a soluble TRAIL decoy receptor respectively. DiFi cells express a detectable level of Fas but are not susceptible to apoptosis induced by the Fas-agonistic mAb CH-11. The mAb 225-induced activation of caspase-8 and apoptosis in DiFi cells was not inhibited by the TNFR1-antagonistic mAb, the soluble TRAIL decoy receptor or a Fas-antagonistic mAb (ZB-4). These results indicate that mAb 225 does not directly activate or regulate these known pathways to activate caspase-8 and induce apoptosis in DiFi cells, and thus suggest the existence of novel mechanism by which mAb 225 triggers apoptosis in DiFi cells.
A human-mouse chimeric version of mAb 225 (C225) is currently being evaluated in clinical trials for its antitumor activity against a variety of human cancers. To achieve maximal cytocidal effect, C225 is being combined with conventional therapies such as chemotherapy and radiation therapy for cancer treatment. The combination therapy has been successful in eradicating well-established xenografts (Baselga et al., 1993; Ciardiello et al., 1999; Fan et al., 1993a; Milas et al., 2000), and current clinical trials with C225 are demonstrating an apparent synergy between mAb C225 and conventional chemotherapy or radiation therapy in patients (Baselga et al., 2000; Mendelsohn et al., 1999; Perez-Soler et al., 1998). Because novel therapies that specifically target relevant molecules in cancer cells and have less toxicity to normal cells are apparently preferable, elucidation of the underlying mechanism by which mAb 225 induces apoptosis in DiFi cells may direct us to develop such novel therapies that, when combined with the use of C225, may reproduce the ‘DiFi-specific’ apoptosis by C225 in a variety of human cancers.
Activation of caspase-8 is essential in inducing apoptosis by mAb 225 in DiFi cells
We have recently shown that the apoptosis induced by the anti-EGF receptor mAb 225 in DiFi cells is characterized by elevated catalytic activity of caspase-8 (Liu et al., 2000). In the current study, we treated three cancer cell lines (A431, MDA468 and DiFi) with mAb 225 and examined for caspase-8 activation. Although all three cell lines express very high levels of EGF receptor, only in DiFi cells was caspase-8 activated following mAb 225 treatment (Figure 1a). This result is consistent with our previous observation that exposure of A431 or MDA468 cells to mAb 225 inhibited cell proliferation (Fan et al., 1993b), whereas exposure of DiFi cells to mAb 225 induced apoptosis (Wu et al., 1995). The levels of caspase-8 expression in these three cell lines following mAb 225 treatment, as measured by Western blot analysis, are shown in Figure 1b. No detectable level of caspase-8 was found in the A431 cells. Caspase-8 was detectable in MDA468 cells but the level was unchanged following treatment. In contrast, there was a time-dependent reduction in the level of caspase-8 in DiFi cells, indicating cleavage and activation of caspase-8 by mAb 225. The caspase-8 doublet in Figure 1b represents two isoforms of caspase-8 (55 and 54 kDa).
We have also recently shown that caspase-9 was also activated in DiFi cells following treatment with mAb 225 (Liu et al., 2000). To determine which caspase (caspase-8 or -9) was essential in mediating mAb 225-induced apoptosis, we examined whether inhibition of caspases-8 or -9 with the caspase-8-specific inhibitor z-IETD-fmk or with the caspase-9-specific inhibitor z-LEHD-fmk would affect mAb 225-induced apoptosis in these cells. Exposure of the cells to z-IETD-fmk or z-LEHD-fmk inhibited the enzymatic activity of caspase-8 or -9, respectively, that was induced by mAb 225 treatment (Figure 2a,b). The inhibition of caspases-8 and -9 enzymatic activities by z-IETD-fmk and z-LEHD-fmk, respectively, was further confirmed by Western blot analysis showing reductions in mAb 225-induced caspase-8 cleavage (activation) by z-IETD-fmk (Figure 2a inset, lane 3 versus lane 4) and in mAb 225-induced caspase-9 cleavage (activation) by z-LEHD-fmk (Figure 2b inset, lane 3 versus lane 4). Figure 2c,d show the effect of z-IETD-fmk or z-LEHD-fmk on mAb 225-induced apoptosis in DiFi cells. Both an apoptosis-specific ELISA (Figure 2c) and a classic DNA agarose gel electrophoresis (Figure 2d) demonstrated that only pretreatment of the cells with the caspase-8 inhibitor z-IETD-fmk inhibited mAb 225-induced apoptosis in the DiFi cells. Pretreatment of the cells with the caspase-9 inhibitor z-LEHD-fmk had no effect on mAb 225-induced apoptosis. Taken together, our data suggest that caspase-8, rather than caspase-9, plays an essential role in mAb 225-induced apoptosis in DiFi cells.
Activation of caspase-8 by mAb 225 is Fas-, TNFR1- and TRAIL-DR4/DR5-independent
Because caspase-8 is activated primarily by the TNF-family death receptor (such as TNFR1, Fas or TRAIL-DR4/DR5)-mediated pathways, we examined whether mAb 225 activated caspase-8 by regulating one of these known pathways. A Fas-antagonistic antibody (ZB-4), a TNFR1 blocking antibody or a decoy TRAIL-R4/DcR2 individually inhibited the Fas-agonistic antibody CH-11, TNFα or TRAIL-induced activation, respectively, of caspase-8 in two positive control cell lines, Jurkat human T-leukemic cells and H460 human lung carcinoma cells. We examined whether these Fas, TNFR1 or TRAIL-DR4/DR5 antagonists could inhibit mAb 225-induced activation of caspase-8 in DiFi cells. Figure 3a presents the results of a caspase-8 enzymatic assay showing that the mAb 225-induced caspase-8 activity (columns 2–5) was not affected by the Fas-antagonistic antibody ZB-4, the anti-TNFR1 antibody or the TRAIL decoy receptor (TRAIL-R4/DcR2), although the TNFR1 blocking antibody and the decoy TRAIL-R4/DcR2 inhibited TNFα- or TRAIL-induced caspase-8 enzymatic activity (column 8 versus column 9 and column 10 versus column 11), respectively, in DiFi cells. The Fas-agonistic antibody CH-11 did not activate caspase-8 in the DiFi cells (column 6), and thus there was no difference in caspase-8 activity in the presence of the Fas-antagonistic antibody ZB-4 (column 7).
The Western blot data (shown in Figure 3b) further confirmed the results of the enzymatic assay of caspase-8 activity (shown in Figure 3a). The reduction (cleavage that leads to activation) in the level of caspase-8 by mAb 225 (lanes 2–5) was not affected by the anti-TNFR1 blocking antibody, the TRAIL decoy receptor (TRAIL-R4/DcR2) or the Fas-antagonistic antibody ZB-4. Consistent with results for the enzymatic assays (Figure 3a), TNFR1 blocking antibody and decoy TRAIL-R4/DcR2 individually reversed the activation of caspase-8 by TNFα (lane 8 versus lane 9) or TRAIL (lane 10 versus lane 11) in DiFi cells. The Fas-agonistic antibody CH-11 had no effect on caspase-8 activity in the presence or absence of the Fas-antagonistic antibody ZB-4 (lane 6 versus lane 7).
The Fas-mediated cell death pathway is not functional in DiFi cells
We next examined whether the Fas-mediated cell death pathway was functional in DiFi cells. We used, as positive control cells, Jurkat cells, which are known to be sensitive to Fas ligand or agonistic mAb treatment. Compared with the results for the Jurkat cells, DiFi cells expressed no detectable level of Fas ligand. The cells constitutively expressed Fas protein, but the level was unchanged by mAb 225 treatment (Figure 4a). As expected, exposure of the Jurkat cells to the Fas-agonistic mAb CH-11 induced apoptosis (measured by an apoptosis ELISA) (Figure 4b, column 2), which was inhibited by the Fas-antagonistic mAb ZB-4 (Figure 4b, column 3). In contrast, exposure of the DiFi cells to the Fas-agonistic mAb did not trigger apoptosis in the cells (Figure 4b, column 5), suggesting that the Fas pathway is not functional in DiFi cells. Treatment of DiFi cells with mAb 225 strongly induced apoptosis (Figure 4b, column 7). This mAb 225-induced apoptosis was not inhibited by the Fas-antagonistic mAb ZB-4 (Figure 4b, column 8). Taken together, these results suggest that the Fas-mediated pathway is not functional and is not involved in mAb 225-induced apoptosis in DiFi cells.
MAb 225-induced apoptosis does not involve TNF receptor-1
To examine whether other pathways that are known to activate caspase-8 were involved in the mAb 225-induced apoptosis, we examined possible involvement of TNFα and TNFR1-mediated cell death pathway in mAb 225-induced apoptosis in DiFi cells (Figure 5). Compared with H460 cells, DiFi cells expressed a very low level of TNFR1 protein (Figure 5a), and treatment of the cells with mAb 225 did not increase the expression of TNFR1. As expected, exposure of H460 cells to TNFα induced apoptosis, as measured by an apoptosis ELISA (Figure 5b column 2). TNFα similarly induced apoptosis in DiFi cells (Figure 5b, column 5), although the extent of apoptosis was less compared with that induced by mAb 225 in the DiFi cells (Figure 5b, column 7). A TNFR1 antibody that blocks the binding of TNFα to the receptor blocked TNFα-induced apoptosis in both H460 cells (Figure 5b, column 2 versus column 3) and DiFi cells (Figure 5b, column 5 versus column 6). However, the TNFR1 blocking antibody did not block mAb 225-induced apoptosis in DiFi cells (Figure 5b, column 7 versus column 8). These results suggest that mAb 225 does not interact with or regulate TNFR1 to induce apoptosis in DiFi cells.
MAb 225 does not interact with or regulate the TRAIL death receptor-mediated cell death pathway
Another important cell death pathway that involves activation of caspase-8 is the TRAIL-induced cell death pathway. There are two types of TRAIL receptor-1 (TRAIL-R1, also known as DR4) and TRAIL receptor-2 (TRAIL-R2, also known as DR5), to which TRAIL binds and thereby activates caspase-8 (Ashkenazi and Dixit, 1998). We thus examined whether the TRAIL pathway was involved in mAb 225-induced apoptosis in DiFi cells. We again used H460 lung carcinoma cells as positive controls, because they have functional TRAIL-R1/DR4 and TRAIL-R2/DR5 pathways (Sun et al., 2001). Compared with H460 cells, DiFi cells expressed similar levels of TRAIL, TRAIL-R1/DR4 and TRAIL-R2/DR5 proteins (Figure 6a). Treatment of DiFi cells with mAb 225 did not change the expression level of either TRAIL or TRAIL receptors. Exposure of H460 cells and DiFi cells to TRAIL induced apoptosis in both cell lines, as measured by apoptosis ELISA (Figure 6b, columns 2 and 5). The TRAIL-induced apoptosis was blocked in both cell lines (Figure 6b, column 2 versus column 3 and column 5 versus column 6) by a soluble TRAIL decoy receptor (TRAIL-R4/DcR2), which contains a normal extracellular domain of the TRAIL receptor but a truncated intracellular domain and thus is unable to transduce death signals (Ashkenazi and Dixit, 1998). In contrast, the mAb 225-induced apoptosis in DiFi cells was not blocked by the decoy TRAIL receptor (Figure 6b, column 7 versus column 8). These results suggest that mAb 225 did not activate or regulate these TRAIL receptors (DR4 and DR5) to induce apoptosis in DiFi cells.
In our current study, we extended our previous investigation of the roles of caspases-8 and -9 in apoptosis induced in DiFi cells by the anti-EGF receptor mAb 225. We here demonstrated that activation of caspase-8 is essential in the induction of apoptosis by mAb 225 and we ruled out the possibility that mAb 225 might directly interact with or regulate the members of human TNF receptor family (such as TNFR1, Fas or TRAIL death receptors) to induce apoptosis in DiFi cells.
Caspase-9 plays a pivotal role in initiating the mitochondrial pathway of apoptosis. Caspase-9 is directly activated by cytochrome c that is released from mitochondria following stimulation of cells with various apoptotic stimuli. Additionally, caspase-9 can also be activated as a consequence of caspase-8 activation. Activated caspase-8 can cleave Bid (a BH3 domain-containing proapoptotic Bcl-2 family member), and the truncated Bid will then translocate to the mitochondria and induce the release of cytochrome c, and the latter will subsequently activate caspase-9 (Li et al., 1998). Therefore, the mAb 225-induced activation of caspase-9 in DiFi cells could result from the activation of caspase-8 by mAb 225 in the cells.
Caspase-8, which was originally termed as FLICE (FADD-like ICE) (Muzio et al., 1996) or MACH (MORT1-associated CED-3 homolog) (Boldin et al., 1996), is the most proximal caspase in the apoptotic pathway triggered by the TNF-family death receptors. These death receptors link via their cytosolic death domains (DD) to the DD of adaptor proteins such as Fas-associated protein (FADD, also known as MORT1) (Boldin et al., 1995; Chinnaiyan et al., 1995), which in turn bind via their death effector domains (DEDs) to caspase-8 (Nagata, 1997; Smith et al., 1994). The current model of caspase-8 activation is as follows: binding of Fas ligand or Fas-agonistic mAb induces trimerization of Fas, which recruits caspase-8 via FADD. The trimerization of caspase-8 results in self-activation of its proteolytic activity through cleavage of its C-terminal CED3/ICE homolog into p20 and p10 fragments. Activated caspase-8 then cleaves and activates downstream effector caspases, such as caspases-3, -6 and -7, which in turn cleave various proteins, including the DFF/ICAD family of endonucleases (Liu et al., 1997; Sakahira et al., 1998).
The activation of caspase-8 and the characteristic morphological change in the induction of apoptosis by mAb 225 in DiFi cells (Liu et al., 2000) are reminiscent of the apoptosis induced by Fas ligand or agonistic antibody in several experimental systems (Itoh et al., 1991). Because mAb 225 is an antibody that binds to cell surface proteins (EGF receptors), it was reasonable to speculate whether mAb 225 crossreacted with one of the members of the death receptor family to trigger apoptosis or whether mAb 225 regulated the functional expressions of death receptors that are known to induce apoptosis in DiFi cells. We recently addressed this speculation by examining the effects of a panel of different anti-EGF receptor mAbs, each of which binds to a distinct epitope on the EGF receptor, on the induction of apoptosis (Liu et al., 2000). In the current study, we examined the effects of blocking several known death receptors on mAb 225-induced apoptosis in DiFi cells. Our data showed that mAb 225 neither crossreacted with nor regulated the functional expression of known death receptors to induce apoptosis in DiFi cells. Our results indicate that the EGF receptor is the primary site at which mAb 225 binds and subsequently induces apoptosis in DiFi cells. However, it is important to note that exclusion of the involvement of TNF receptor family members in mAb 225-induced activation of caspase-8 and the subsequent induction of apoptosis does not rule out the possible involvement of FADD/MORT1 in activating caspase-8 by mAb 225.
Similar instances of caspase-8 activation by means that were independent of Fas- or other TNF family death receptors have been reported in apoptosis induced by ultraviolet irradiation (Sheikh et al., 1998), ionizing radiation (Belka et al., 1999), cytotoxic drugs (Bantel et al., 1999; Beltinger et al., 1999; Micheau et al., 1999a; Wesselborg et al., 1999) and interferon (Balachandran et al., 2000; Gil and Esteban, 2000) as well as in anoikis (detachment-induced apoptosis) (Rytomaa et al., 1999). The death receptor-independent activation of caspase-8 in these studies was either FADD-dependent (Balachandran et al., 2000; Beltinger et al., 1999; Gil and Esteban, 2000; Micheau et al., 1999b; Rytomaa et al., 1999; Sheikh et al., 1998) or FADD-independent (Bantel et al., 1999; Wesselborg et al., 1999). We are currently investigating whether the mAb 225-induced activation of caspase-8 requires FADD/MORT1 or alternatively uses a FADD-like adaptor protein.
In our study, we found that both TNFα and TRAIL activated caspase-8 and induced apoptosis in DiFi cells. However, although DiFi cells expressed a fair amount of Fas, the cells were not susceptible to Fas-agonistic mAb-induced apoptosis. The reasons why the Fas-agonistic mAb CH-11 did not activate caspase-8 and failed to induce apoptosis in the DiFi cells were not further explored in the current study, because this would deviate from our focus. Previous studies in literature have shown that the failure of cells that express Fas to undergo apoptosis following treatment with either Fas ligand or Fas-agonistic mAb could be due to either the expression of FAP-1 (Fas-associated phosphatase-1), which can block the apoptotic function of Fas (Li et al., 2000; Ungefroren et al., 1998) or the secretion by certain types of tumors of a soluble Fas that can protect Fas-sensitive cells from Fas ligand- or Fas-agonistic mAb-induced apoptosis (Fellenberg et al., 1997).
In conclusion, our results suggest that mAb 225 does not interact with or regulate the TNF family receptor members to activate caspase-8 and induce apoptosis. Thus, an exploration is strongly warranted for a novel mechanism by which mAb 225 triggers apoptosis in DiFi cells.
Materials and methods
The anti-EGF receptor mAb 225 has been described previously (Fan et al., 1993b, 1994; Kawamoto et al., 1983; Sato et al., 1983). Recombinant human TNFα was purchased from Knoll Pharmaceutics (Whippany, NJ, USA). Recombinant soluble TRAIL was from Biomal Research Laboratories, Inc. (Plymouth Meeting, PA, USA). The Fas-agonistic mAb clone CH-11 and the Fas neutralization mAb clone ZB-4 were from Upstate Biotechnology, Inc. (Lake Placid, NY, USA) and MBL International Corp. (Watertown, MA, USA), respectively. The human TNFR1 blocking mAb clone 16803.1 was from R&D System, Inc. (Minneapolis, MN, USA). Recombinant TRAIL-R4/DcR2, caspase-8 substrate Ac-IETD, pNA, and caspase-9 substrate Ac-LEHD-pNA were from Alexis Corp. (San Diego, CA, USA). Caspase-8 specific inhibitor z-IETD-fmk and caspase-9 specific inhibitor z-LEHD-fmk were from Enzyme Systems Products (Livermore, CA, USA).
Antibodies for Western blot analysis were from following sources: caspase-8 mAb (clone 5F7) (Upstate Biotechnology, Inc.); caspase-9 polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA); Fas mAb (clone G254-274), Fas ligand mAb (clone G247-4), TRAIL mAb (clone B35-1) and TRAIL-R1/DR4 polyclonal antibody (PharMingen Biotechnology, Inc., San Diego, CA, USA); TRAIL-R2/DR5 polyclonal antibody (Cayman Chemical Co., Ann Arbor, MI, USA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified.
Cells and cell culture
DiFi human colorectal adenocarcinoma cells, A431 squamous carcinoma cells, and MDA468 breast carcinoma cells were described previously (Liu et al., 2000, 2001; Wu et al., 1995, 1996). The H460 human non-small cell lung carcinoma line was provided by Dr Shi-Yong Sun (The University of Texas MD Anderson Cancer Center, Houston, TX, USA). The aforementioned cells were maintained in a mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (1 : 1, v/v) supplemented with 10% fetal bovine serum (FBS). Jurkat human T leukemic cells were obtained from Dr Gordon B Mills (MD Anderson Cancer Center) and were maintained in RPMI 1640 medium supplemented with 10% FBS. All the cell lines were cultured in a 37°C humidified atmosphere containing 95% air and 5% CO2 and were split twice a week.
Caspase enzymatic activity assay
Caspase enzymatic activities were measured by using a colorimetric assay kit from Clontech Laboratories, Inc. (Palo Alto, CA, USA) as we previously reported (Liu et al., 2000). The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA), which is cleaved from caspase-specific substrates by activated caspases (IETD-pNA by activated caspase-8, and LEHD-pNA by activated caspase-9).
Quantification of apoptosis
An apoptosis ELISA kit (Roche Diagnostics Corp., Indianapolis, IN, USA) was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) as we previously reported (Liu et al., 2000). This photometric enzyme immunoassay was performed exactly according to the manufacturer's instructions.
Analysis of DNA fragmentation
The method for demonstration of internucleosomal DNA fragmentation has been described previously (Wu et al., 1995). Cells (approximately 1×106) were washed with cold PBS solution and then lysed with 200 μl of lysis buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% mg/ml proteinase K. The lysates were incubated in a 50°C water bath for 1 h prior to the addition of heat-boiled RNaseA to a final concentration of 0.5 mg/ml; lysates were then incubated for an additional 1 h in the 50°C water bath. Samples were then diluted with an equal volume of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). DNA was extracted three times with phenol-chloroform-isoamyl alcohol (25 : 24 : 1) and precipitated overnight with two volumes of ethyl alcohol (−20°C) and 0.2 volume of 3 M ammonium acetate. The DNA precipitates were dissolved in TE buffer. DNA (5 μg) was loaded on a 1.5% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA) for electrophoresis. The agarose gel was stained with ethidium bromide, and the resulting DNA fragmentation was visualized by ultraviolet illumination and photographed.
Western blotting analysis
Cells were lysed in an NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin, 25 μg/ml aprotinin) and sonicated at 4°C. The supernatants were cleared by centrifugation. Protein concentrations were measured by using the Coomassie Plus protein assay reagent (Pierce Chemical Co., Rockford, IL, USA). Equal amounts of cell extracts were boiled in Laemmli SDS-sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with different primary antibodies. After the blots were incubated for 1 h at room temperature with horseradish peroxidase-labeled secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG, Bio-Rad Laboratories, Hercules, CA, USA), the signals were detected using the Enhanced Chemiluminescence (ECL) assay (Amersham Life Science Inc., Arlington Heights, IL, USA), according to the manufacturer's instructions.
epidermal growth factor
transforming growth factor-α
cysteinyl aspartate-specific proteinase
tumor necrosis factor-α
TNF-related apoptosis inducing ligand
enzyme-linked immunosorbent assay
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The authors are grateful to Dr John Mendelsohn for support and encouragement of this study, to Dr Shi-Yong Sun of the Department of Head and Neck Oncology for many stimulating discussions, and to Mr Michael Worley of the Department of Scientific Publications for editorial assistance with the manuscript. This study was supported in part by the NCI cancer center core grant (CA16672), by a Bristol-Myers Squibb Research Award, and by a start-up fund to Z Fan by The University of Texas MD Anderson Cancer Center.
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