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[Cancer Research 64, 3922-3927, June 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Oncogenic Ras Sensitizes Normal Human Cells to Tumor Necrosis Factor-{alpha}-Related Apoptosis-Inducing Ligand-Induced Apoptosis

Alexandre Nesterov, Malti Nikrad, Thomas Johnson and Andrew S. Kraft

Division of Medical Oncology, University of Colorado Health Sciences Center, Denver, Colorado


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces apoptosis in tumor cells but rarely kills normal ones. To determine how normal human cells acquire TRAIL-sensitive phenotype during the process of malignant transformation, we used an experimental system that allows for controlled conversion of human cells from normal to cancerous by introduction of several genes. Human embryonic kidney cells and foreskin fibroblasts were first immortalized by combination of the early region of simian virus 40 and telomerase and then were transformed with oncogenic Ras. Both normal and immortalized cells were resistant to TRAIL-induced apoptosis, whereas Ras-transformed cells were susceptible. Ras transformation enhanced TRAIL-induced activation of caspase 8 by increasing its recruitment to TRAIL receptors. The proapoptotic effects of Ras could be reversed by mutations in its effector loop or by inhibitors of either farnesyl transferase or mitogen-activated protein kinase kinase. The expression of constitutively activated mitogen-activated protein kinase kinase 1 enhanced caspase 8 recruitment and sensitized immortalized human embryonic kidney cells to TRAIL-induced death. These results indicate that in normal human cells the TRAIL-induced apoptotic signal is blocked at the level of caspase 8 recruitment and that this block can be eliminated by Ras transformation, involving activation of the mitogen-activated protein kinase pathway.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that selectively induces apoptosis in a variety of tumor cells but rarely affects normal ones (1) . At least five receptors for TRAIL have been identified. Two of these, DR4 (TRAIL-R1) and DR5 (TRAIL-R2), contain a conserved motif, the death domain, and signal apoptosis (2) . Binding of TRAIL to DR4 or DR5 induces formation of the death-inducing signaling complex (DISC), which comprises the adaptor protein Fas-associated death domain (FADD)/MORT1 and the FADD-binding cysteine protease, caspase 8/FLICE (3 , 4) . The formation of the DISC triggers proteolytic autoprocessing and activation of caspase 8, which in turn cleaves and activates the downstream caspases 3, 6, or 7, leading to apoptosis. In addition, caspase 8 cleaves a cytolinker, plectin, which induces reorganization of the cytoskeleton (5) , and the proapoptotic protein, Bid, which generates a cleavage product that triggers the release of cytochrome c from mitochondria (6 , 7) .

The reason that TRAIL is selective toward cancer cells despite the ubiquitous expression of its receptors in normal tissues remains unclear. Malignant transformation is believed to require stepwise accumulation of at least three genetic alterations: inactivation of tumor suppressors, immortalization, and receipt of a continuous mitogenic signal (8) . To investigate at which step of this process and how human cells acquire a TRAIL-sensitive phenotype, we used a recently developed experimental system that mimics stepwise progression of human cells from normal to tumorigenic (9) . Normal cells were converted to tumorigenic by serial introduction of the early region of simian virus (SV40ER) to disable tumor suppressors p53 and Rb, a catalytic subunit of telomerase (hTERT) to ensure their unlimited life span, and an oncogenic allele of Ha-Ras (H-ras-V12) to provide cells with a continuous growth signal. On transformation with activated Ras, these cells become capable of anchorage-independent growth and formation of tumors in immunodeficient mice (9) . This model allowed us to investigate a mechanism by which malignant transformation renders human cells susceptible to TRAIL-induced apoptosis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
The following reagents were obtained from the indicated sources: SCH 66336 (Schering-Plough Research Institute); PD 98059 and U0126 (Calbiochem); z-VAD-FMK (Enzyme System Products); anti-phospho-mitogen-activated protein kinase (anti-phospho-ERK) and anti-ERK (New England Biolabs); anti-Bid (Zymed Laboratories); antiplectin (Transduction Laboratories); anti-caspase 8 and anti-FADD (Upstate Biotechnology); monoclonal antibody to caspase 10 (Clone 4C1; MBL International); anti-DR4 and anti-DR5 (Alexis); monoclonal antibody NF6 to FLIP (a gift of Marcus Peter, Ben May Institute for Cancer Research). The expression and purification of TRAIL from yeast Pichia pastoris has been described in detail elsewhere (10) .

Cell Culture.
Normal human foreskin fibroblasts (BJ) were obtained from the American Type Culture Collection. Normal human embryonic kidney (HEK) cells were kindly provided by Silvia Bacchetti (McMaster University). Immortalization of the cells with SV40 large T antigen and hTERT and their subsequent transformation with H-ras-V12 has been described in detail elsewhere (9) . Different Ras and mitogen-activated protein kinase kinase (MEK) constructs were expressed in immortalized HEK cells by use of pBabe-Puro vector (Ref. 11 ; a gift of Scott W. Lowe, Cold Spring Harbor Laboratory). Retroviral stocks were generated in Phoenix ecotropic packaging line (G. Nolan, Stanford University), and stable transformants were selected in the presence of puromycin (500 ng/ml). Cells infected with an empty vector were used as a control. Human bladder cancer cell line T24 was kindly provided by Gary J. Miller (University of Colorado Health Sciences Center, Denver, CO).

Cytotoxicity Assays.
Cell viability was determined by either the tetrazolium-based Aqueous One assay (Promega) or by staining with 7-aminoactinomycin D and flow cytometry. Detection of DNA fragmentation by agarose gel electrophoresis was performed with a Suicide-Track DNA Ladder Isolation Kit (Oncogene Research Products), using a procedure that selectively extracts apoptotic DNA from intact chromatin.

DISC Immunoprecipitation.
HEK cells grown in roller bottles (~4 x 108 cells/condition) were scraped into 10 ml of conditioned medium, combined, precipitated, and resuspended in 5 ml of the conditioned medium. As judged by the trypan blue exclusion assay, >80% of cells remained viable after this procedure. Stimulation with TRAIL was achieved by incubation with TRAIL (1 µg/ml) for 20 min at 37°C. The cells were washed in ice-cold PBS, lysed in 10 ml of Triton/glycerol/HEPES buffer (12) , cleared by centrifugation, and equalized for protein content. Precipitation of the unstimulated TRAIL receptors was achieved by adding TRAIL (1 µg/ml) to the lysates for 30 min at 4°C. The TRAIL receptors that were complexed with TRAIL were immunoprecipitated by addition of 25 µl of antipolyhistidine agarose (Sigma) for 2 h at 4°C, and bound proteins were eluted with 100 mM glycine-HCl (pH 2.3; two times 40 µl for 10 min at 4°C).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Conversion of Normal Human Cells to Tumorigenic Ones Sensitizes Them to TRAIL.
HEK and BJ cells were first immortalized by the combination of SV40ER and hTERT and then transformed with oncogenic Ras (9) . Both normal and immortalized cells remained resistant to TRAIL, whereas the introduction of activated Ras rendered them susceptible to TRAIL-induced apoptosis, as assessed by both morphological changes typical of apoptosis (Fig. 1A)Citation and DNA fragmentation (Fig. 1B)Citation .



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  		Fig. 1. Transformation of human embryonic kidney cells (HEK) and human foreskin fibroblasts (BJ) with Ras sensitizes them to tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The effects of TRAIL (1 µg/ml) on normal (N), immortalized (I), or Ras-transformed (R) HEK cells and BJ fibroblasts were assessed 72 h after the addition of TRAIL. A, the morphology of the plated cells was assessed, and representative photographs are shown. B, the cells were harvested, and extracts were processed by DNA laddering assay. DNA laddering was visualized by ethidium bromide staining of 2% agarose gels. Molecular weight markers are shown on the right.

 
Ras Transformation Potentiates TRAIL-Induced Activation of the Initiator Caspase 8.
Because the process of immortalization did not sensitize cells to TRAIL-induced apoptosis, these cells were used as the control for additional experiments. To determine what step in the TRAIL-mediated apoptotic cascade is enhanced by Ras transformation, we compared TRAIL-induced proteolytic events in Ras-transformed and control, immortalized cells. As shown in Fig. 2Citation , the addition of TRAIL to control cells did not induce cleavage of the initiator caspase 8. In contrast, treatment of Ras-transformed cells with TRAIL induced significant caspase 8 cleavage. Interestingly, TRAIL-induced processing of another proximal caspase, caspase 10 (13) , was not facilitated by Ras transformation (Fig. 2)Citation , suggesting that these two enzymes may be regulated in different ways.



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  		Fig. 2. Transformation of cells with Ras enhances the ability of tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL) to activate caspase 8. Control (Cont) cells (immortalized cells infected with empty vector) or Ras-transformed human embryonic kidney cells (HEK) and human foreskin fibroblasts (BJ) were treated for 72 h with 1 µg/ml TRAIL. Cell lysates were immunoblotted with antibodies specific for caspase 8, caspase 10, Bid, and plectin. Equal protein loading was confirmed by probing the blot with antibodies to Fas-associated death domain (FADD).

 
Caspase 8 cleavage is not necessarily accompanied by an increase in the activity of this proteolytic enzyme toward its cellular substrates (14) . We therefore examined the ability of Ras to enhance TRAIL-induced cleavage of two specific caspase 8 substrates, Bid (6 , 7) and plectin (5) . As shown in Fig. 2Citation , oncogenic Ras enhanced the ability of TRAIL to induce the cleavage of plectin in both HEK and BJ cells. Although we could not detect significant cleavage of Bid in BJ cells, proteolytic processing of this protein was evident in Ras-transformed HEK cells, which were generally more responsive to TRAIL than BJ fibroblasts.

Ras Transformation Enhances Recruitment of Caspase 8 to TRAIL DISC.
We next examined how transformation of cells with Ras affected the ability of TRAIL receptors to form a functional DISC. To immunoprecipitate the activated TRAIL receptors DR4 and DR5, we first incubated intact cells with a (His)6-tagged recombinant TRAIL (10) and then immunoprecipitated the receptors with an antipolyhistidine antibody. To immunoprecipitate unstimulated receptors, we first lysed cells with detergent and then added (His)6-TRAIL to the extracts.

These and subsequent experiments were performed with the HEK cells because the proapoptotic effects of Ras transformation were more robust in these cells. As shown in Fig. 3Citation , the antibody to (His)6-TRAIL efficiently immunoprecipitated both TRAIL receptors, DR4 and DR5. The lack of immunoprecipitation of TRAIL receptors when the cells or cell lysates were not treated with (His)6-TRAIL confirmed the specificity of this procedure. DR5 was detected as a doublet, corresponding to two known splice variants of this protein (15) . This result demonstrates that oncogenic Ras does not significantly affect the level of DR4 in these cells but elevates the expression of DR5. Densitometric analysis of two independent experiments revealed the 1.5–2-fold increase of DR5 expression in Ras-transformed versus control cells.



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  		Fig. 3. Transformation of cells with Ras enhances the recruitment of caspase 8 to Fas-associated death domain (FADD). Control and Ras-transformed human embryonic kidney cells (HEK) were treated with (His)6-tagged tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (1 µg/ml) for 20 min at 37°C (TRAIL before lysis). The cells were lysed, and TRAIL–TRAIL-receptor complexes were immunoprecipitated (IP) with monoclonal antibody to polyhistidine. To immunoprecipitate unstimulated receptors, TRAIL was added to cells after lysis. Aliquots of the immunoprecipitated material were analyzed by immunoblotting with antibodies specific for DR5, DR4, FADD, and caspase 8. DR5 is detected as a doublet, corresponding to the two different splice variants of this protein (15) . Equal amounts of protein in cell lysates was confirmed by probing with antibodies to FADD and caspase 8.

 
When cells were treated with TRAIL before lysis, two additional proteins, FADD and caspase 8, coimmunoprecipitated with the TRAIL receptors (Fig. 3)Citation . FADD coimmunoprecipitated equivalently with TRAIL receptors from Ras-transformed and control cells. In contrast, caspase 8 was recruited to TRAIL receptors in Ras-transformed cells much more efficiently than in control cells. Densitometric analysis of two independent experiments demonstrated that Ras increased caspase 8 recruitment >=8-fold. This result suggests that Ras transformation sensitizes cells to TRAIL by facilitating the binding of caspase 8 to FADD.

To rule out the possibility that the observed difference in the amounts of coimmunoprecipitated caspase 8 resulted from different rates of caspase 8 processing and subsequent dissociation from the DISC, cells were pretreated with the pancaspase inhibitor Z-VAD-FMK. Inhibition of caspase 8 activity, however, did not have a significant effect on the amounts of caspase 8 bound to the TRAIL receptors (Fig. 3)Citation .

The Proapoptotic Effects of Ras Are Reversible.
To test whether sensitization of cells to TRAIL by Ras transformation is a reversible process, we used the farnesyl transferase inhibitor SCH 66336, a compound that inhibits prenylation of Ras proteins and suppresses their biological activity (16) . As shown in Fig. 4, A and BCitation , pretreatment of Ras-transformed HEK cells with SCH 66336 efficiently rescued them from TRAIL-induced death.



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  		Fig. 4. Effects of small molecule inhibitors on tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of Ras-transformed cells. A and B, Ras-transformed human embryonic kidney (HEK) cells were pretreated for 72 h with farnesyl transferase inhibitor SCH 66336 (1 µM), mitogen-activated protein (MEK) inhibitors PD 98059 (100 µM) and U0126 (20 µM), or 0.1% DMSO as a control. Cells were then treated for an additional 72 h with different concentrations of TRAIL. A, the relative viability of cells was evaluated by use of the tetrazolium conversion assay. B, the cells were harvested, and extracts were processed by DNA laddering assay. DNA laddering was visualized by ethidium bromide staining of 2% agarose gels. C, Ras-transformed HEK cells were pretreated with one of the small molecule inhibitors as described above and then incubated with TRAIL (1 µg/ml) for 48 h. After this treatment, the cells were lysed and immunoblotted with an antibody to caspase 8. A short exposure of the electrochemiluminescence-treated blot was used to visualize holocaspase 8, and a long exposure was used for its 18-kDa proteolytic fragment. D, T24 human bladder cancer cells were pretreated for 72 h with farnesyl transferase inhibitor SCH 66336 (1 µM), MEK inhibitor PD 98059 (100 µM), or 0.1% DMSO as a control. Where indicated, cells were then treated for an additional 24 h with TRAIL (1 µg/ml). After this treatment, the cells were lysed and immunoblotted with an antibody to caspase 8.

 
To test whether the Ras-induced MAP kinase pathway contributes to the proapoptotic effect of this oncogene, we used two inhibitors of MEK, PD 98059 (17) and U0126 (18) . As shown in Fig. 4, A and BCitation , the MEK inhibitors efficiently rescued Ras-transformed HEK cells from TRAIL. Both the MEK and farnesyl transferase inhibitors suppressed TRAIL-induced cleavage of caspase 8 (Fig. 4C)Citation . These compounds thus appear to specifically reverse the proapoptotic effects of Ras.

We next tested whether Ras-dependent sensitization of cells to TRAIL-induced apoptosis can also occur in transformed cells obtained from cancer patients. For this purpose we used the bladder cancer cell line T24, which expresses the oncogenic allele of Ha-Ras (H-ras-V12) and possesses constitutively activated MAP kinase (19) . Using the farnesyl transferase inhibitor SCH 66336 or the MEK inhibitor PD 98059, we found that inhibition of either Ras processing or MAP kinase activity suppressed TRAIL-induced caspase 8 cleavage in T24 cells (Fig. 4D)Citation and rescued them from TRAIL-mediated apoptosis (data not shown). This result confirms that sensitization of cells to TRAIL by oncogenic Ras can indeed take place in spontaneous human cancers.

The Proapoptotic Effects of Ras Are Mediated by MAP Kinase Pathway.
The results presented in Fig. 4Citation suggested that the proapoptotic effects of Ras may involve the MAP kinase pathway. To confirm this observation, we transformed immortalized HEK cells with retroviruses expressing either a gain-of-function mutant of MEK1 (MEK1Q56P), a dual-specificity protein kinase that phosphorylates and activates MAP kinase (11 , 20) , or two effector loop mutants of Ras (RasV12C40 and Ras V12G37) that are defective for Raf binding and do not activate the MAP kinase pathway (21, 22, 23) . As measured by incorporation of 7-aminoactinomycin D, TRAIL efficiently induced death in cells that expressed either RasV12 or MEK1Q56P but not in cells expressing constructs defective in MAP kinase activation (Fig. 5)Citation . These results confirm that an activated MAP kinase pathway is essential for sensitization of HEK cells to TRAIL-induced apoptosis.



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  		Fig. 5. Proapoptotic effects of Ras are mediated by the mitogen-activated protein kinase pathway. Immortalized human embryonic kidney (HEK) cells were infected with empty retrovirus (EV), retroviruses expressing different Ras constructs (as indicated), activated mutant of mitogen-activated protein kinase 1 (MEK1Q56P) or wild-type MEK1 [MEK(WT)]. Uninfected cells were used as control (Cont). The cells were treated with 1 µg/ml tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL) for 48 h. Apoptotic cells were detected by incorporation of 7-aminoactinomycin D (7AAD). Data are presented as percentage of 7AAD-positive cells after subtraction of 7AAD uptake by cells not treated with TRAIL. The data shown are the average of three determinations and the SD (bars) of the mean.

 
Triggering of MAP Kinase Pathway Enhances TRAIL-Induced Activation of Caspase 8.
The results presented in Figs. 2Citation and 3Citation suggest that oncogenic Ras sensitizes cells to TRAIL-inducing apoptosis by enhancing activation of caspase 8. To investigate the involvement of the MAP kinase pathway in this process, we used a constitutively activated mutant of MEK1, MEK1Q56P. Using an antibody that specifically recognizes the phosphorylated, activated form of MAP kinase, we found that expression of MEK1Q56P induced phosphorylation of MAP kinase to approximately the same level as that observed in Ras-transformed cells (Fig. 6A)Citation .



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  		Fig. 6. Activation of the mitogen-activated protein (MAP) kinase pathway potentiates tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL)-induced recruitment of caspase 8. Human embryonic kidney (HEK) cells were infected by a retrovirus expressing constitutively the activated mutant of MAP kinase kinase (MEK), MEK1Q56P. A, control cells (C), MEK1Q56P-expressing cells (M), or Ras-transformed cells (R) were lysed and analyzed by immunoblotting with antibodies specific for phosphorylated MAP kinase (phospho-ERK) or total MAP kinase (ERK). B, control, MEK1Q56P-expressing (MEK), or Ras-transformed cells were treated for 48 h with TRAIL (1 µg/ml). Cell lysates were immunoblotted with antibodies specific for caspase 8, FLIP, and Bid. Equal protein loading was confirmed by probing the blot with antibodies to Fas-associated death domain (FADD). C, upper panel, where indicated, immortalized HEK cells were exposed to UV irradiation at 100 µJ/cm2 and then treated with TRAIL (1 µg/ml) for 3 h. Cell lysates were subjected to Western blotting with an antibody to caspase-8. Lower panel, cells were irradiated as described above, followed by incubation for various times. Cell lysates were subjected to Western blotting with an antibody to phosphorylated ERK (phospho-ERK). D, TRAIL receptors were immunoprecipitated with the antibody to polyhistidine from control (C), Ras-transformed (R), or MEK1Q56P-expressing cells (M) as described in the legend to Fig. 3ACitation . Where indicated, cultures were pretreated for 72 h with farnesyl transferase inhibitor SCH 66336 (1 µM) or MEK inhibitor PD 98059 (100 µM). Aliquots of the immunoprecipitated materials were analyzed by immunoblotting with antibodies specific for DR4, DR5, caspase 8, and Fas-associated death domain (FADD).

 
As demonstrated in Fig. 6BCitation , activation of the MAP kinase pathway potentiates the same step in the TRAIL-induced apoptotic cascade as oncogenic Ras. Treatment of either Ras-transformed or MEK1Q56P-expressing, but not control, cells with TRAIL induced cleavage of both caspase 8 and two caspase 8 substrates, cFLIPL and Bid.

We also tested whether TRAIL-induced activation of caspase 8 can be enhanced by some stress stimuli known to trigger the MAP kinase pathway. For this purpose we treated HEK cells with UV light, a potent activator of the ERK pathway (24) . As demonstrated in Fig. 6CCitation , UV irradiation resulted in greatly increased caspase 8 processing in response to TRAIL.

The results presented in Fig. 3Citation suggested that oncogenic Ras sensitizes cells to TRAIL by enhancing the recruitment of caspase 8 to TRAIL DISC. To confirm that this effect is mediated by the MAP kinase pathway, we analyzed DISC formation under conditions in which this pathway was either inhibited by PD 98059, the MEK inhibitor, or activated by the expression of MEK1Q56P. TRAIL–TRAIL-receptor complexes were immunoprecipitated from TRAIL-treated cells by use of an antibody to polyhistidine. As shown in Fig. 6DCitation , comparable amounts of FADD coimmunoprecipitated with TRAIL receptors from control, Ras-transformed, or MEK1Q56P-expressing cells. In contrast, significantly more caspase 8 was coimmunoprecipitated with TRAIL receptors from Ras-transformed cells than from control cells. Pretreatment of Ras-transformed cells with the farnesyl transferase inhibitor SCH 66336 or with the MEK inhibitor PD 98059 reduced the amounts of coimmunoprecipitated caspase 8. The expression of constitutively active MEK increased the recruitment of caspase 8 to TRAIL receptors almost as efficiently as did oncogenic Ras. These results indicate that the observed enhancement of TRAIL receptor DISC formation in Ras-transformed cells is mediated by the MAP kinase pathway.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although selective killing of cancer cells by TRAIL has been reported in many studies (1) , the molecular mechanisms behind this selectivity remain unclear. Research has been focused on a comparison of apoptotic signals induced by TRAIL in normal cells versus those obtained from cancer patients. These studies implicated several proteins, including the protease-deficient homolog of caspase 8, FLIP (25) , and the TRAIL decoy receptors DcR1 and DcR2 (26) , in resistance of normal cells to TRAIL. However, a firm correlation between the cellular expression of these proteins and TRAIL resistance has not been established (27 , 28) .

Given the number of genetic alterations that occur during neoplastic transformation in humans (8) , it is difficult to single out individual changes that are responsible for the acquisition of the TRAIL-sensitive phenotype. Here we describe the use of genetically defined transformation to investigate how conversion of human cells from normal to tumorigenic renders them sensitive to TRAIL-induced apoptosis. This experimental model mimics the stepwise accumulation of genetic alterations that occurs in human cancers: inactivation of tumor suppressors, acquisition of an unlimited life span, and receipt of a continuous mitogenic signal (9) . Both normal and immortalized cells were found to be resistant to TRAIL-mediated apoptosis, indicating that premalignant changes, including inactivation of tumor suppressors and acquisition of an unlimited life span, are not sufficient to sensitize cells to TRAIL. However, the subsequent conversion of immortalized cells to tumorigenic ones by activated Ras rendered them susceptible to TRAIL-mediated apoptosis.

Ras-mediated sensitization of cells to TRAIL-induced apoptosis may involve at least two mechanisms. First, oncogenic Ras up-regulates the levels of one of the TRAIL receptors, DR5. Second, transformation of cells with Ras appears to facilitate recruitment of caspase 8 to TRAIL DISC. Because the overall amounts of FADD associated with TRAIL receptors was not increased on Ras transformation, it is likely that activated Ras somehow facilitates the interaction of caspase 8 with FADD. This process may involve several mechanisms. For example, one can speculate that the Ras-induced MAP kinase pathway may trigger direct phosphorylation/dephosphorylation of caspase 8, thereby affecting its FADD-binding function. Alternatively, the MAP kinase pathway may down-regulate the expression of a protein that competes with caspase 8 for FADD binding. Obviously, additional studies are needed to determine the exact mechanism of this sensitization.

Because Ras is capable of inducing genomic instability (29) , it might, in principle, sensitize cells to TRAIL by promoting irreversible genetic alterations that would disable certain antiapoptotic pathways. Two observations make this scenario unlikely. First, the Ras-transformed cells used in our studies were found to be polyclonal (9) . Second, the proapoptotic effects of Ras could be reversed by inhibitors of either farnesyl transferase or MEK. Apparently, continuous Ras signaling is essential for Ras-transformed cells to maintain both the transformed phenotype (30) and sensitivity to TRAIL. Alternatively, the activity of the farnesylation inhibitor could be related to its ability to regulate RhoB (31) , suggesting that regulation of other small GTP-binding proteins may be important in regulating the sensitivity to TRAIL.

Our results indicate that the proapoptotic effect of Ras is mediated by the MAP kinase pathway. It has been reported recently that activation of this pathway suppresses TRAIL-induced apoptosis in HeLa cells (32) . One possible explanation for this apparent discrepancy is that MAP kinase is capable of eliciting both proapoptotic (24 , 33 , 34) and prosurvival responses (35) . The antiapoptotic effect of MAP kinase appears to be cell type specific. For example, activation of MAP kinase by phorbol myristate acetate was shown to suppress receptor-mediated apoptosis in some, but not all, types of cells (36) . A prosurvival effect of the MAP kinase pathway has, at least in part, been attributed to ERK-dependent up-regulation of antiapoptotic FLIP proteins (37) . However, positive regulation of FLIP expression by the MAP kinase pathway has been observed only in a limited set of human cells (38) . Likewise, we did not observe any significant changes in the levels of FLIP proteins either on activation of MAP kinase by Ras or constitutively activated MEK (Fig. 6B)Citation , or on treatment of Ras-transformed BJ and HEK cells with MEK inhibitors (data not shown).

In summary, using human cells that were progressively converted from normal into tumorigenic we demonstrated that (a) premalignant changes, including inactivation of tumor suppressors and immortalization, are not sufficient to sensitize human cells to TRAIL; (b), transformation of the immortalized cells with the growth-promoting oncogene H-ras-V12 renders them susceptible to TRAIL-induced apoptosis; (c) oncogenic Ras potentiates TRAIL-induced recruitment and activation of the initiator caspase 8; (d) the proapoptotic effects of Ras are reversible and involve the MAP kinase pathway; and (e) constitutive activation of MAP kinase sensitizes immortalized human cells to TRAIL. Because aberrant activation of MAP kinases is often associated with a neoplastic phenotype (19) , sustained MAP kinase activity may potentially serve as an indicator of malignant transformation recognized by a TRAIL-based antitumor surveillance system.


   ACKNOWLEDGMENTS
 
We thank Drs. William C. Hahn and Robert A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA) for immortalized and Ras-transformed HEK and BJ cells; Dr. Scott W. Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) for the MEK and Ras constructs; Dr. Silvia Bacchetti (McMaster University, Hamilton, Ontario, Canada) for normal HEK cells; Dr. W. Robert Bishop (Schering-Plough Research Institute, Kenilworth, NJ) for the farnesyl transferase inhibitor, SCH 66336; Dr. Marcus E. Peter (Ben May Institute for Cancer Research, Chicago, IL) for anti-FLIP antibody NF6; and Dr. Gary Nolan (Stanford University, Palo Alto, CA) for the Phoenix packaging cell line.


   FOOTNOTES
 
Grant support: Department of Defense Grant DAMD 17-01-1-0045 (to A. S. Kraft).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Andrew E. Kraft, Division of Medical Oncology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail: Andrew.Kraft{at}UCHSC.edu

Received 7/21/03. Revised 2/27/04. Accepted 3/16/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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