Apoptosis of Melanoma Cells (TRAIL)-Induced

The Role of NF-κB in TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis of Melanoma Cells This information is current as of July 18, 2013.

Agustin V. Franco, Xu Dong Zhang, Elisabeth Van Berkel, Jayne E. Sanders, Xi Yi Zhang, Wayne D. Thomas, Tam Nguyen and Peter Hersey J Immunol 2001; 166:5337-5345; ; http://www.jimmunol.org/content/166/9/5337

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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References

The Role of NF-␬B in TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis of Melanoma Cells1 Agustin V. Franco, Xu Dong Zhang, Elisabeth Van Berkel, Jayne E. Sanders, Xi Yi Zhang, Wayne D. Thomas, Tam Nguyen, and Peter Hersey2

S

everal members of the TNF family have been shown to induce apoptosis in susceptible cells by activation of the caspase pathways (1–3). Induction of apoptosis appears to be restricted to receptors that contain “death domains” such as those for Fas (CD95) ligand (FasL).3 TNF-␣, TNF-related apoptosis-inducing ligand (TRAIL)/apo-2 (4, 5), and apo-3 ligand (apo3L; Ref. 6). TRAIL appears to be particularly important, as it appears to be able to induce apoptosis in a wide range of neoplastic but not normal cells (4). TRAIL can induce apoptosis by interaction with two receptors referred to as DR4 (TRAIL-R1) (7, 8) and DR5/TRAIL-R2 (9 –11). These receptors were found to be widely expressed on normal tissues, but the latter are believed to be protected from apoptosis by two additional receptors, TRAIL-R3/ DcR1 (12–14) and TRAIL-R4/DcR2 (15, 16). The latter are believed to inhibit apoptosis either by competition for binding of TRAIL (e.g., acting as decoys) or by providing signals that inhibit apoptosis (3, 17). Activation of the transcription factor NF-␬B may be one of the main pathways involved in the latter, as it is known to be activated by TRAIL-R4 (17) and, paradoxically, also by death receptors for TRAIL (8). NF-␬B is known to regulate the expression of several proteins that inhibit apoptosis, such as inDepartment of Oncology and Immunology Unit, David Maddison Clinical Sciences Building, Newcastle, New South Wales, Australia Received for publication April 12, 2000. Accepted for publication February 16, 2001. 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.

hibitors of apoptosis (XIAP, IAP1, and IAP2) that block apoptosis by inhibition of caspase-8 (18) and caspase-3 and -9 (19) and the protein A1, which is a Bcl-2 homologue (20). Evidence for the importance of activation of NF-␬B in resistance to apoptosis came from studies on leukemia cells in which inhibition of NF-␬B was associated with increased sensitivity to apoptosis induced by TNF-␣ and activation of CD95 and TRAIL (21). TRAIL-induced apoptosis of keratinocyte lines was inhibited by IL-1, which was shown to activate NF-␬B (22). Some melanoma cells were shown to become sensitive to TNF-␣ when they were transfected with a dominant-negative form of I␬B, which inhibited NF-␬B activation (23). Similarly, inhibition of NF-␬B was found to sensitize chemoresistant tumors to TNF-␣ and chemotherapy (24) and to reduce the in vivo survival of human head and neck squamous cell carcinomas (25). We have shown previously that TRAIL (26 –28) but not other members of the TNF family (29, 30) can induce apoptosis in approximately two-thirds of melanoma cell lines. There was marked variability in response to TRAIL, which in some lines was related to loss of TRAIL receptor expression (27) but in other lines, the basis for resistance to TRAIL was not clear. In the present study, we have assessed the role of NF-␬B in susceptibility of melanoma cells to apoptosis induced by TRAIL and other members of the TNF family by use of agents which inhibit NF-␬B activity. We also have generated TRAIL-resistant melanoma cells by culture in TRAIL and show that activation of NF-␬B appears to be a key transcription factor involved in resistance to TRAIL but not other TNF family members.

1 This work supported by the Melanoma and Skin Cancer Research Institute, Sydney, Australia, and New South Wales State Cancer Council, New South Wales, Australia. 2 Address correspondence and reprint requests to Dr. Peter Hersey, Department of Oncology and Immunology, Room 443, David Maddison Building, Corner of King and Watt Streets, Newcastle, NSW 2300, Australia. E-mail address: Peter.Hersey@ newcastle.edu.au 3

Abbreviations used in this paper: FasL, Fas ligand; TRAIL, TNF-related apoptosisinducing ligand; TRAIL-R, receptors for TRAIL; hu, human; DcR, decoy receptor for TRAIL; DR, death receptor for TRAIL; GFP, green fluorescent protein; LLnL, Nacetyl-Leu-Leu-norleucinal; IAP, inhibitors of apoptosis. Copyright © 2001 by The American Association of Immunologists

Materials and Methods Cell lines Melanoma cell lines with the prefix Mel were isolated from fresh surgical biopsies from patients attending the Sydney and Newcastle Melanoma Units and established in the laboratory. FH, CV, AT, and GL were from lymph nodes. MC was from skin. RM, JG, and SP were from bowel. The cell lines had been in culture for 2– 6 mo at the time of these studies. 0022-1767/01/$02.00


Previous studies have shown that activation of NF-␬B can inhibit apoptosis induced by a number of stimuli. It is also known that TNF-related apoptosis-inducing ligand (TRAIL) can activate NF-␬B through the death receptors TRAIL-R1 and TRAIL-R2, and decoy receptor TRAIL-R4. In view of these findings, we have investigated the extent to which activation of NF-␬B may account for the variable responses of melanoma lines to apoptosis induced by TRAIL and other TNF family members. Pretreatment of the melanoma lines with the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL), which is known to inhibit activation of NF-␬B, was shown to markedly increase apoptosis in 10 of 12 melanoma lines with death receptors for TRAIL. The specificity of results for inhibition of NF-␬B activation was supported by an increase of TRAIL-induced apoptosis in melanoma cells transfected with a degradation-resistant I␬B␣. Furthermore, studies with NF-␬B reporter constructs revealed that the resistance of melanoma lines to TRAIL-induced apoptosis was correlated to activation of NF-␬B in response to TRAIL. TRAIL-resistant sublines that were generated by intermittent exposure to TRAIL were shown to have high levels of activated NF-␬B, and resistance to TRAIL could be reversed by LLnL and by the superrepressor form of I␬B␣. Therefore, these results suggest that activation of NF-␬B by TRAIL plays an important role in resistance of melanoma cells to TRAIL-induced apoptosis and further suggest that inhibitors of NF-␬B may be useful adjuncts in clinical use of TRAIL against melanoma. The Journal of Immunology, 2001, 166: 5337–5345.

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RESISTANCE OF MELANOMA CELLS TO TRAIL-INDUCED APOPTOSIS

MM200, Me1007, Me10538, Me4405, and IgR3 were from primary melanoma. The derivation of MM200, Me1007, Me10538, and Me4405, are described elsewhere (27, 30). SK-MEL-110 and SK-MEL-28 were a kind gift from Dr. A. Albino (American Health Foundation, Valhalla, NY) and S. Ralph (Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia), respectively. All melanoma cell lines were positive for tyrosinase and MART-1 mRNA by RT-PCR tests described elsewhere (31), except for Mel-SP, which was positive for Tyrosinase but not MART-1. All cell lines were cultured in DMEM containing 5% FCS (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia).

CA) for 24 h. Fluorescence was measured by flow cytometry, as mentioned in Materials and Methods. To determine the efficiency of transfection, the cells were cotransfected with 30 ␮g of a vector encoding the LacZ cDNA, and percentage of blue cells were estimated 24 h after transfection. In some experiments, the mutant I␬B␣-containing vector at 30 ␮g DNA/2,000,000 cells in 400 ␮l was cotransfected with pEG FP-N1GFP vector (Clontech) at 1.5 ␮g of DNA. Control cultures were transfected with the GFP vector at 30 ␮g of DNA/2,000,000 cells.

Plasmid vectors and transfectants

Recombinant human (hu) TRAIL (lot 6321-19) prepared as described elsewhere (4) and huCD40L (lot 5753-56) were supplied by Immunex (Seattle, WA). Each preparation was supplied as a leucine zipper fusion protein, which required no further cross-linking for maximal activity. Recombinant huFasL, produced from isolated cDNA (GenBank accession no. U08137) in vector pDC409 and transfected into COS cells, was kindly supplied as sterile supernatants by Immunex. It produced 50% lysis of Jurkat T cells at dilutions of 2– 4 (30). rTNF-␣ cytokines and control mAb antitrinitrophenyl (anti-TNP; IgG1) were purchased from BD PharMingen (Bioclone, Marrickville, Australia). Control mAb 1D4.5 (Ig2a) was against an Ag on Salmonellae (kindly supplied by L. Ashman, Hansen Cancer Research Centre, Adelaide, Australia). The MAbs against TRAIL-R1 (IgG2a huTR1-M271; lot: 7136-07), TRAIL-R2 (IgG1 huTRAIL-R2-M413; lot: 5274-96), TRAIL-R3 (IgG1 huTR3-M430; lot: 7313-217), and TRAIL-R4 (IgG1 huTR4-M444; lot: 7136-15) were supplied by Immunex and are described elsewhere (33). The rabbit Abs against p65 were raised against the C-terminal end peptide SGDEDFSSIADMDFSALLSQIS and anti-p75 against the C-terminal end peptide MMTTSSDSMGETDNPRLLSM, and were purchased from Stressgen (Victoria, British Columbia, Canada). The rabbit Ab against p50 was purchased from Fitzgerald (Concord, MA). The control purified rabbit IgG (prod. no. 15006) was purchased from Sigma (St. Louis, MO). The proteasome inhibitor (calpain inhibitor 1) N-acetylLeu-Leu-norleucinal (LLnL) (34) was purchased from Sigma (cat. no. Ab2185). Lactacystin (35) was purchased from ICN Biochemicals (Biomedicals Australasia, Seven Hills, New South Wales, Australia).

Flow cytometry Analysis was conducted with a Facscan flow cytometer (Becton Dickinson, Mountain View, CA) . In studies on relocalization of TRAIL receptors, melanoma cells were grown overnight in 24-well plates and exposed to TRAIL 100 ng/ml for 30 min. Adherent cells were removed by trypsinization in 0.25% trypsin at 37°C for 5 min, washed twice in cold DMEM and once in PBS at 4°C, and fixed in 4% paraformaldehyde. Appropriate concentrations of mAbs were added to the cells in 100 ␮l of PBS containing 20% human A serum and incubated for 7 min at room temperature. Cells were washed either twice with PBS and analyzed if directly labeled, or if indirectly labeled, cells the were incubated with F(ab⬘)2 affinity-isolated FITC-conjugated sheep anti-mouse Ig (Silenus, cat. no. 985051020; Amrad Biotech, Boronia, Victoria, Australia) plus 20 ␮l of 100% human serum to

FIGURE 1. Gel shift assays of NF-␬B activation status in seven melanoma lines before and after exposure to TRAIL 100 ng/ml for 30 min and with or without LLnL at 5 ␮M for 2 h before addition of TRAIL. TRAIL increased activation of NF-␬B, and this was reduced in the presence of LLnL. Five lines (Mel-CV, MM200, Mel-RM, SK-MEL-28, and Mel-FH) had constitutive activation of NF-␬B.

FIGURE 2. A, Kinetics of activation of NF-␬B in Mel-FH by TRAIL (100 ng/ml) showing activation by 5 min after exposure to TRAIL. Tracts are as follows: 1) Jurkat; 2) Jurkat ⫹ TRAIL; 3) Mel-FH; 4) Mel-FH ⫹ TRAIL for 5 min; 5) Mel-FH ⫹ TRAIL for 15 min; 6) Mel-FH ⫹ TRAIL for 30 min; 7) Mel-FH ⫹ TRAIL for 120 min; 8) Mel-FH ⫹ TRAIL in presence of unlabeled NF-␬B oligonucleotide (negative control). B, Dose response for TRAIL-induced activation of NF-␬B. Tracts are as follows: 1) Jurkat; 2) Jurkat ⫹ TRAIL 100 ng/ml; 3) Mel-FH; 4) Mel-FH ⫹ TRAIL 0.1 ng; 5) Mel-FH ⫹ TRAIL 1 ng/ml; 6) Mel-FH ⫹ TRAIL 10 ng/ml; 7) Mel-FH ⫹ TRAIL 50 ng/ml; 8) Mel-FH ⫹ TRAIL 100 ng/ml.


A superrepressor form of human I␬B␣ protein S32A/36A resistant to degradation (in the vector PRC/CMV) was kindly provided by Steve Gerondakis (Walter and Eliza Hall Institute, Melbourne Victoria, Australia) (32). Stable and transient transfectants were conducted by electroporation by a Gene Pulser (Bio-Rad, Hercules, CA) as follows: MM200, 0.36 kV and 960 ␮F; Mel-RM, 0.33 kV and 500 ␮F; Mel-FH, 0.36 kV and 500 ␮F; SK-MEL-28, 0.32 kV and 500 ␮F; Mel-CV, 0.36 kV and 500 ␮F; and Me4405, 0.30 kV and 500 ␮F. Twenty-four hours after electroporation, G418 was added to a final concentration of 400 ␮g/ml for 21 days or until colonies appeared on the plate. For the analysis of NF-␬B activation in transient transfectants, the cells were cotransfected with 30 ␮g of pNF␬B-d2EGFP, a reporter vector encoding the green fluorescent protein (GFP) under the control of the ␬ enhancer element (Clontech, Palo Alto,

Monoclonal Abs, recombinant proteins, and peptides

The Journal of Immunology

5339

Table I. Increase in sensitivity of melanoma cell lines to TRAIL-induced apoptosis in the presence of an inhibitor of NF-␬B, LLnL Concentration of LLnL (␮M) Cell Line

0

– ⫹ – ⫹ – ⫹ – ⫹ – ⫹ – ⫹ – ⫹ – ⫹ – ⫹

0 0 0 3 0 13 0 45 0 32 0 0 0 0 0 0.6 0 3.7

Me1007 Mel-AT Mel-CV MM200 Mel-FH Melanocytes PBL Activated PBLb Fibroblasts (MRC-5)

2.5

12 21 (9)a 9 15 (3) 1 41 (27) 1 56 (16) 1 45 (12) 0 0 0 0 0.2 2.5 0.5 4.3

5

62 69 (7) ND 4 42 (25) 3 67 (25) 4 56 (20) 0 0 0 0 0.9 10.0 0.7 3.8

7.5

66 68 (2) 21 32 (8) 10 47 (24) 6 68 (23) 15 57 (10) 0 0 0 0 – – 1.0 2.8

a Figures in parentheses indicate increase in percentage of apoptosis above that induced by TRAIL alone less apoptosis induced by LLnL alone. b Activated by exposure to anti-CD3 for 24 h.

block Fc receptors for 7 min at room temperature. A minimum of 5000 cells was analyzed. Studies on permeabilized cells were similar to the methods of Jung et al. (36). The cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% saponin in permeabilization buffer and the Ab added for 30 min at 4°C. The cells were washed and then stained by FITC-labeled F(ab)2 fraction of affinity-isolated sheep anti-mouse Ig (Silenus; Amrad Biotech) at 1/100 dilution for 30 min at 4°C. After washing, the cells were analyzed by flow cytometry. The TRAIL-R-negative Me10538 cell line (27) was included as a negative control in studies on permeabilized cells. The percentage of cells expressing the receptors was calculated as the difference in positive area between the positive and negative control histograms. The positive area was that to the right of the intersection of the two curves (37). For the detection of nuclear localization of NF-␬B proteins P50, P65, and P75, the melanoma cells were stained by Cycletest PLUS DNA reagent kit (Becton Dickinson, Lane Cove, Australia) as described elsewhere (38). The cells were resuspended in 5 ml of citrate buffer and centrifuged at 300 ⫻ g for 5 min. The supernatant was discarded and the cells resuspended in 1.5 ml of citrate buffer and centrifuged for a further 5 min at

Table II. Increase in sensitivity of melanoma cells to TRAIL-induced apoptosis resulting from pretreatment with the proteasome inhibitor LLnL Percentage of Apoptosis (%)

Cell Line

TRAIL (100 ng/ml)

LLnL (5 ␮M)

TRAIL ⫹ LLnLa

Mel-AT Me1007 Mel-MC Me4405 Mel-GL Mel-RM IgR3 Mel-CV Mel-FH MM200 SK-MEL28 Mel-JG Mel-SP Me10538

3 3 14 35 31 29 21 12 30 48 18 80 0 0

15 62 26 14 14 16 5 4 7 3 23 6 0 2

24 69 60 61 73 78 79 42 52 67 91 84 0 2

a The figures in bold indicate levels of apoptosis 5% or above that from the sum of apoptosis induced by TRAIL or LLnL alone.

300 ⫻ g. The supernatant was carefully discarded and the cells blotted dry. The pellet was gently resuspended in a mixture of 250 ␮l of solution A (trypsin in a spermine tetrahydrochloride detergent buffer) and 200 ␮l of solution B (trypsin inhibitor and RNase A in citrate stabilizing buffer with spermine tetrahydrochloride) for 10 min at room temperature. Abs against P65, P50, or P75 then were added for 10 min at room temperature, followed by incubation for 10 min with 2.5 ␮l of FITC-conjugated anti-mouse mAb (Amrad Biotech). Percentage of expression of the proteins was calculated as above for receptor expression.

Measurement of apoptosis Apoptotic cells were determined by the propidium iodide method (39). In brief, melanoma cells were adhered overnight in a 24-well plate (Falcon 3047; Becton Dickinson, Lane Cove, Australia) at a concentration of 1 ⫻ 105/well in 10% FCS. Cells in suspension were added on the day of the assay. Medium was removed and cells treated for 20 h with the reagents TRAIL, huFasL, TNF-␣, or huCD40L. Cells were incubated for a further 24 h at 37°C, the medium removed, and adherent and suspended cells washed 1 ⫻ with PBS. The medium and PBS were placed in 12 ⫻ 75 mm Falcon polystyrene tube and centrifuged at 200 ⫻ g. A total of 1 ml of hypotonic buffer (propidium iodide, 50 ␮g/ml, in 0.1% sodium citrate plus 0.1% Triton X-100; Sigma) was added directly to the cell pellet of cells grown in suspension or to adhered cells in the 24-well plate and gently pipetted off, then added to the appropriate cell pellet in the Falcon tube. The tubes were placed at 4°C in the dark overnight before flow cytometric analyses. The propidium iodide fluorescence of individual nuclei was measured in the red fluorescence with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and the data registered in a logarithmic scale. At least 104 cells of each sample were analyzed. Apoptotic nuclei appeared as a broad hypodiploid DNA peak, which was easily distinguished from the narrow hyperdiploid peak of nuclei in the melanoma cells. In cotransfection experiments with the GFP vector, apoptosis was assessed in cells gated for green fluorescence by annexin V conjugated with PE (BD PharMingen). TRAIL 100 ng/ml was added ⬃48 h after transfection and apoptosis measured 3 h later.

EMSA For the preparation of whole-cell lysates, melanoma cells (2 ⫻ 106) were lysed in 80 ␮l of 600 mM KCl, 20 mM HEPES, pH 7.05, 200 ␮M EDTA, 1 mM PMSF, 10 ␮g/ml soybean trypsin inhibitor, and 2 ␮g/ml aprotinin for 45 min on ice (all obtained from Sigma) and centrifuged at 12,000 ⫻ g for 5 min at 4°C. For the preparation of cytoplasmic and nuclear extracts from melanomas, we followed the methods described by Dignam et al. (40). Cells (5 ⫻ 106) were washed twice with ice-cold 1⫻ PBS and spun down at 500 ⫻ g at 4°C for 5 min. The cell pellet was subsequently


TRAIL (100 ng/ml)

5340


Table III. The proteasome inhibitor LLnL does not increase apoptosis in melanoma cells induced by other TNF family membersa % TNF-R Cell Line

Mel-CV Mel-FH MM200 SK-MEL-28 Me10538 Mel-JG a b

LLnL

2 7 3 3 2 2

TRAIL

11 20 48 23 0 73

LLnL ⫹ TRAIL b

66 46 70 47 2 86

FasL

LLnL ⫹ FasL

% Fas

3 1 2 0 19 2

10 7 9 2 21 5

83 34 20 8 6 0

TNF-␣

LLnL ⫹ TNF-␣

⫺1

⫺2

CD40L

LLnL ⫹ CD40L

% CD40

0 1 0 0 4 0

8 6 4 3 6 2

21 17 12 9 9 2

18 0 11 0 11 0

0 0 0 0 0 0

5 8 3 0 3 2

63 9 12 13 8 0

LLnL was used at 2.5 ␮M, TRAIL and TNF-␣ at 100 ng/ml, and CD40L at 300 ng/ml. FasL was used at a dilution of 1:100. The figures in bold indicate levels of apoptosis 5% or above the sum of apoptosis due to LLnL or TRAIL alone.

Results

Inhibition of TRAIL-induced activation of NF-␬B by LLnL The proteasome inhibitor LLnL was shown in previous studies to inhibit the degradation of I␬B and thereby to prevent activation of NF-␬B (21, 34). To determine whether LLnL inhibited activation of NF-␬B in melanoma cells, we used EMSA to measure NF-␬B activation in the melanoma lines before and after activation by TRAIL and the effect of LLnL on this activation. As shown in Fig. 1, enhanced NF-␬B binding activity induced by TRAIL was shown in seven of the melanoma lines selected for study. Constitutive NF-␬B activation was found in five cell lines, SK-MEL-28, MelCV, Mel-FH, MM200, and Mel-RM, before addition of TRAIL (Fig. 1). LLnL (5 ␮M) added 2 h before TRAIL (and throughout culture with TRAIL) reduced the binding to barely detectable levels, except in extracts from Me4405 that still had relatively high levels of activated NF-␬B after pretreatment with LLnL. The level of NF-␬B activation shown in the gel shift assays were consistent with the levels shown by NF-␬B reporter assays shown below. As shown in Fig. 2A, NF-␬B activation occurred within 5 min and was

maximal from 15 to 60 min after exposure to TRAIL. NF-␬B activation was maximal at a concentration of 10 ng/ml (Fig. 2B). This is less than the optimal concentration of 100 ng/ml shown for TRAIL-induced apoptosis (26). Similar results were found in studies on the Mel-CV line. The proteasome inhibitor LLnL that inhibits activation of NF-␬B can reverse resistance to TRAIL-induced apoptosis We then examined whether LLnL would inhibit TRAIL-induced apoptosis of melanoma cells in studies on a large number of melanoma lines. The dose titration studies shown in Table I indicate that two of the lines, Me1007 and Mel-AT, showed marked sensitivity to LLnL alone, and there was only a small increase in apoptosis induced by TRAIL in the presence of LLnL. Three melanoma lines that were partially sensitive to TRAIL-induced apoptosis had more marked increases in sensitivity to TRAIL in the presence of LLnL that approached maximum levels in the assay. It also was noted that treatment of melanocytes, fibroblasts, and resting PBL with even high concentrations of LLnL did not make them sensitive to TRAIL-induced apoptosis. However, there was a small increase in sensitivity of PBL activated by anti-CD3 to TRAIL-induced apoptosis. These studies were repeated at a fixed concentration of LLnL (5 ␮M) in a wider panel of melanoma cells, as shown in Table II. Again, there was wide variation in toxicity of LLnL between lines but in most lines there was a marked increase in TRAIL-induced apoptosis in the presence of LLnL, the exceptions being the control cell lines Me10538 and Mel-SP, which did not have death receptors for TRAIL (27). The TRAIL-sensitive line Mel-JG was maximally killed by TRAIL alone, and no further increase was induced by LLnL. Studies with a second proteasome inhibitor, lactacystin (35), at 10 ␮M on the cell lines Me1007, Mel-CV, Mel-FH, and MM200 gave a similar increase in TRAIL-induced apoptosis (data not shown). Inhibition of NF-␬B with LLnL has minimal effects on the level of apoptosis induced by other TNF family members

FIGURE 3. Gel shift assay of NF-␬B activation status in two melanoma lines showing activation of NF-␬B in SK-MEL-28 cells by CD40L, FasL, and TRAIL, and in Mel-FH cells by FasL and TRAIL. TRAIL 100 ng/ml was added 30 min before the assay.

In view of the increased sensitivity of melanoma cells to TRAILinduced apoptosis in melanoma cells exposed to LLnL, we also examined whether activation of NF-␬B may be involved in resistance of melanoma cells to TNF-␣, FasL, and CD40L. Six cell lines that showed a wide range of sensitivity to TRAIL-induced apoptosis were pretreated with LLnL 3 h before addition of TRAIL, FasL, TNF-␣, and CD40L in optimum concentrations established in previous studies (26, 29, 30). The results shown in Table III indicate that LLnL did not sensitize the melanoma cells to apoptosis induced by TNF-␣ or CD40L but did potentiate low levels of apoptosis induced by FasL in the cell line Mel-CV. In contrast, LLnL increased apoptosis induced by TRAIL in all but the control TRAIL-R-negative line Me10538. It was of interest that FasL induced


homogenized in 200 ␮l of Dignam buffer A containing protease inhibitors as described above. The homogenate was spun down, and the nuclear pellet was washed twice with buffer A. The nuclear pellet then was resuspended in 30 ␮l of Dignam buffer C. A total of 10 ␮grams of supernatant was incubated with 4 ␮l of 5⫻ gel shift binding buffer (Promega, Madison, WI) for 30 min at room temperature to double-stranded oligonucleotides encoding the consensus binding sites of NF-␬B (5⬘-AGTTGAGGGGACTT TCCCAGGC-3⬘) that had been labeled with [32P]ATP (GeneWorks, Adelaide, Australia) by polynucleotide kinase (Promega). For supershift assays, Abs against P50 and P65 were incubated at room temperature for 30 min with the melanoma lysates in the presence of the radiolabeled oligo. Protein-oligo complexes were separated on a nondenaturing 6% PAGE in 0.5 ⫻ Tris-borate-EDTA buffer. Gels were dried and exposed overnight to an x-ray film. Included in each assay was a positive control of extracts from the Jurkat T cell line exposed to TRAIL and a negative control of unlabeled NF-␬B and random AP2 oligonucleotide included in the Promega NF-␬B gel shift assay system (cat. no. E3300).


5341

FIGURE 4. A, Gel shift assay of activated NF-␬B protein binding in extracts of the cytoplasm and nuclei of Mel-RM. After exposure to TRAIL some degradation and increased mobility of NF-␬B proteins was evident in cytoplasmic extracts but there was, as expected, a marked increase in NF-␬B protein binding in the nucleus after exposure to TRAIL. B, Supershift gel assays of extracts from Mel-RM with Abs against p50 and p65. This confirms that the proteins bound to the probe are NF-␬B proteins.

Cytoplasmic and nuclear location of activated NF-␬B To further substantiate the gel shift assays of NF-␬B activation we examined the site of the activated NF-␬B proteins in the melanoma cells by studies on extracts from the nucleus and cytoplasm of the cells that were prepared as described elsewhere (40). As shown in Fig. 4A, TRAIL induced a typical increase in NF-␬B activation in extracts from the nuclei of Mel-RM cells and there were only relatively small amounts in the cytoplasm. The specificity of the probe for the NF-␬B proteins also was also shown by the supershift electromobility assays with Abs to p50 and p65 NF-␬B proteins, illustrated in Fig. 4B.

The levels of TRAIL-induced activation of NF-␬B measured in reporter assays are higher in TRAIL-resistant compared with TRAIL-sensitive cell lines In view of the variable levels of NF-␬B activation in the melanoma lines before and after exposure to TRAIL shown in Fig. 1, we used more quantitative NF-␬B reporter assays to examine the relation between NF-␬B activation and TRAIL-induced apoptosis. Transient transfection with the NF-␬B reporter vector pNF-␬B-d2EGFP was possible in eight of nine melanoma lines but was not possible in the Me4405 line. The results shown in Table IV have been arranged in order of the sensitivity of the cell lines to TRAIL and %GFP levels have been adjusted to take into account the variable transfection efficiency. The relatively resistant cell lines Mel-FH and Me1007 had low basal NF-␬B levels, but after treatment with TRAIL, the levels of NF-␬B increased by 73 and 166% respectively (Table IV). IgR3, Mel-RM, and SK-MEL-28 had moderate sensitivity to TRAIL and moderately high basal levels of NF-␬B, which was increased further by TRAIL. In contrast, the TRAIL-sensitive lines, Mel-JG and MM200, had high basal levels of NF-␬B that were relatively unchanged after treatment with TRAIL. Mel-CV also had high basal levels and relatively low levels of TRAIL-induced activation of NF-␬B but remained relatively resistant to TRAIL-induced apoptosis. Essentially similar results were found in a repeat of the study, with the exception of Mel-CV. These results

Table IV. Sensitivity of melanoma cells to TRAIL-induced apoptosis appears inversely related to the level of TRAIL-induced NF-␬B activation measured by GFP reporter assays

Cell Line

Mel-JG MM200 IgR3 Mel-RM Mel-FH SK-MEL28 Mel-CV Me1007 a

TRAIL (100 ng/ml)

% Apoptosis

NF-␬B Activation % Expression of GFPa

– ⫹ – ⫹ – ⫹ – ⫹ – ⫹ – ⫹ – ⫹ – ⫹

2.1 85 3.2 61.6 1.6 42 2.2 31.4 3.1 23.0 1.6 17.9 2.6 11.8 0 4

10.2 10.7 54.0 57.8 37 47 33 41 17.7 30.6 16.5 22.3 57.1 66.0 3 8

b

MFI

1.61 1.65 1.39 1.46 2.03 2.29 1.16 1.29 1.1 1.31 1.12 1.21 1.12 1.25 1.1 1.28

Corrected Values % Expression of GFPc

93 97 81 86 69 88 75 93 50.6 87.4 71.7 96.9 84.0 97.0 33 88

% Change Induced by TRAILd

4 6 28 24 73 35 16 166

% Transfection

11 11 67 67 53 53 44 44 35 35 23 23 68 68 9 9

Percentage of cells expressing GFP by flow cytometry. MFI, Median fluorescent intensity. Values corrected for percentage of transfection by dividing by percentage of transfection. The latter was assessed by percentage of blue/blue ⫹ white colonies. d Percentage of change induced by TRAIL was calculated by dividing the increase in GFP by the basal GFP level. b c


low levels of apoptosis in the latter and this was not increased by LLnL. All of the cell lines expressed receptors for the other members of the TNF family except the TRAIL-sensitive line Mel-JG. The results shown are representative of two experiments. To determine whether NF-␬B was activated by other members of the TNF family, NF-␬B activation was measured in gel-shift assays on two of the cell lines (SK-MEL-28 and Mel-FH) that did not undergo apoptosis after exposure to the TNF family members. As shown in Fig. 3, NF-␬B was activated by FasL and TRAIL in both melanoma lines and by CD40L in the SK-MEL-28 line, but NF-␬B activation was not associated with apoptosis. However, the results show that the receptors were expressed at sufficient levels to be functionally active.

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Table V. Inhibition of NF-␬B activation by transfection of dominant negative I␬B␣ is associated with increased apoptosis and decreased binding of NF-␬B proteins to the nucleus and percentage of cells expressing GFP from reporter gene % Staining of Nuclear Proteinsa,b Cell Line

Mel-FH Mel-FH ⫹ vector Mel-FH ⫹ I␬B␣ Mel-RM Mel-RM ⫹ vector Mel-RM ⫹ I␬B␣ a b

% GFP Expression

% Apoptosis Without TRAIL

TRAIL-Induced % Apoptosis

p50

p65

p75

2.2 3.2 4.1 1.6 2.8 3.6

23.2 24.0 52.3 31.4 24.7 50.2

15.8 ⫾ 2.3 26.7 ⫾ 0.6 9.1 27.3 ⫾ 0.4 22.1 ⫾ 0.8 2.3 ⫾ 2.7

18.1 ⫾ 0.8 20.7 ⫾ 1.5 7.6 ⫾ 0.8 36.6 ⫾ 10.7 29.1 ⫾ 11.4 12.2 ⫾ 10.2

15.4 ⫾ 1.4 18.2 ⫾ 1.8 3.1 ⫾ 0.3 21.1 ⫾ 3.3 15.3 ⫾ 5.3 4.2 ⫾ 4.2

No TRAIL

TRAIL

10.0 14.0

23.0 0.0

33.0 37.4

41.0 27.3

Results shown for Mel-FH and Mel-RM are means ⫾ SD from three experiments. Values shown were obtained 30 min after exposure to TRAIL (100 ng/ml).

suggest a reciprocal relation between the TRAIL-induced activation of NF-␬B and TRAIL-induced apoptosis of melanoma. Regression analysis of percentage of apoptosis vs percentage of increase in GFP expression, including all the results in Table IV, showed a significant inverse relationship (y ⫽ 54.0 ⫺ 0.39 ⫻ r2 ⫽ 0.55; p ⫽ 0.03.).

The findings above that TRAIL-induced apoptosis was increased by treatment with LLnL did not prove that the increase was attributable to inhibition of NF-␬B activation, as proteasome inhibitors also inhibit the breakdown of other proteins that may be involved in apoptosis, such as p53 (41). In view of this, we transfected a mutated degradation-resistant form of I␬B␣ into the melanoma cells that specifically inhibits activation of NF-␬B by binding the proteins in the cytoplasm. Transient transfection of the degradation-resistant I␬B␣ was conducted in two cell lines,

Generation of TRAIL-resistant cultures of melanoma cells

FIGURE 5. Melanoma cells were cotransfected with mutated I␬B␣ and a GFP-expressing vector at 1/20 the concentration of DNA, and 48 h later, sensitivity to TRAIL-induced apoptosis was assessed on cells gated for GFP expression by PE-conjugated annexin V 3 h after addition of TRAIL (100 ng/ml). TRAIL-induced apoptosis was more marked in the I␬B␣transfected cells compared with those transfected with the vector alone. Values shown are means ⫾ SEM.

To identify factors associated with resistance to TRAIL more clearly we generated TRAIL-resistant lines by exposure to TRAIL in vitro. Three melanoma cell sublines, Mel-FHSel, MM200Sel, and Mel-RMSel, were grown in the presence of TRAIL. Briefly, the parental cell lines were seeded in 25 cm2 flasks and TRAIL (100 ng/ml) was added. The surviving cells were fed every three days for 3– 6 wk with tissue culture medium containing TRAIL until they reached confluence and were resistant to TRAIL-induced apoptosis. The levels of NF-␬B measured by the NF-␬B GFP reporter construct in the Mel-RM and MM200 TRAIL-selected cells shown in Table VI were higher than the parental cell lines in the study shown in Table IV. Similar results were shown in a repeat of this study. To determine whether resistance to TRAIL-induced apoptosis was related to activation of NF-␬B the in vitro generated TRAILresistant melanoma lines Mel-FHSel, MM200Sel, and Mel-RMSel were pretreated with LLnL (2–5 ␮M) and their sensitivity to TRAIL examined. As shown in Table VIII, there was a marked increase in sensitivity of the TRAIL-resistant lines MM200Sel, Mel-RMSel, and Mel-FHSel to TRAIL in the presence of LLnL that exceeded the level of apoptosis in the parental lines. The results shown are representative of two experiments. Evidence for


A superrepressor form of I␬B␣ increases TRAIL-induced apoptosis in melanoma cells

Mel-FH and Mel-RM. The levels of I␬B␣ in the transfectants were confirmed by FACS with anti-I␬B␣ (data not shown). As shown in Table V, TRAIL-induced apoptosis was increased in each of the melanoma lines to levels comparable to that seen with the proteasome inhibitor LLnL. This was accompanied by decreased binding of p50, p65, and p75 to the nuclei of Mel-FH and Mel-RM, shown by flow cytometry on isolated nuclei. Inhibition of NF-␬B activation also was shown in NF-␬B GFP reporter assays on Mel-RM and Mel-FH in that there was a decrease in %GFP expression after exposure to TRAIL in the I␬B␣-transfected melanoma cells but not in the control cells transfected with the vector alone. To ensure that apoptosis was measured only in cells transfected with the mutant I␬B␣ we cotransfected a GFP-expressing vector at a twentieth of the concentration of the I␬B␣ vector and apoptosis was measured in cells gated for green fluorescence by annexin V conjugated with PE. Given the relatively low concentration of the GFP vector, it was safe to assume the GFP-containing cells also were transfected with the mutant I␬B␣ vector. The results of three studies on the cell lines Mel-RM and Mel-FH shown in Fig. 5 indicate that transfection of the mutant I␬B␣ was associated with a marked increase in apoptosis induced by TRAIL relative to cells transfected with the vector alone and relative to cells not exposed to TRAIL. The higher degree of apoptosis associated with transfection of I␬B␣ in the absence of TRAIL presumably reflects the greater sensitivity of the annexin V assay.


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Table VI. TRAIL-selected resistant melanoma cells express high levels of NF-␬B activation measured in GFP reporter assays

Cell Line

MM200 MM200Sel Mel-RM Mel-RMSel

TRAIL (100 ng/ml)

% Apoptosis

NF-␬B Activation % Expression of GFP

– ⫹ – ⫹ – ⫹ – ⫹

3.2 61.6 2.8 6.5 2.2 31.4 3.0 4.5

54 57.8 67.2 69.0 33 41 39.7 43.6

Corrected Valuesa

81 86 97 100.0 75 93 84.5 89.0

% Change Induced by TRAIL

6 2.5 24 7

% Transfection

67 67 69 69 44 44 47 49

a

Values corrected for percentage of transfection by dividing by percentage of transfection. The latter was assessed by percentage of blue/blue ⫹ white colonies.

Discussion Our previous studies (26 –28) and those by others (33, 42) show that TRAIL appears to be particularly active in induction of apoptosis in melanoma cells whereas other members of the TNF family had little or no such activity (26, 29, 30). Nevertheless, we found previously that there was a large variation in response of different melanoma cell lines to TRAIL. This was accounted for in part by the level of expression of the TRAIL death receptors, R1 and R2, on melanoma cells but some cell lines were resistant to TRAIL despite relatively high levels of TRAIL-R expression (27). A number of studies have shown that TRAIL-R not only activates the caspase pathway leading to apoptosis but also transmits signals that activate the transcription factor NF-␬B (8, 17). This was confirmed in the present studies by gel shift assays, GFP reporter constructs for NF-␬B, and nuclear staining with Abs specific for p50, p65, and p75. NF-␬B is known to activate a number of genes coding for proteins that inhibit apoptosis by the transmembrane signaling pathway (18, 43) and mitochondrial stress pathway (20,

44). To investigate the possible role of NF-␬B in resistance to TRAIL-induced apoptosis, we used an inhibitor, LLnL, that blocks the ability of proteasomes to degrade I␬B. Therefore, I␬B remains bound to NF-␬B in the cytoplasm and prevents its relocation to the nucleus. A mutant form of I␬B␣ that was not degraded by proteasomes also was used to confirm the specificity of the effects for inhibition of the activation of NF-␬B. The results indicated that LLnL reversed the resistance to TRAIL-induced apoptosis in 10 of 12 melanoma lines with death receptors for TRAIL. Two lines did not show an increase in sensitivity to TRAIL in the presence of LLnL. One (Me10538) did not express TRAIL-R (27) and another line, Me1007, was difficult to interpret because it underwent cell death in the presence of LLnL alone. The latter result was similar to studies on Hodgkin’s lymphoma cells that found that proliferation and viability was dependent on activation of NF-␬B in the cells (45). Another line, MelJG, was very sensitive to TRAIL-induced apoptosis alone, and LLnL could not further increase this sensitivity. These results suggest that NF-␬B activation is a significant factor in protection against TRAIL-induced apoptosis of melanoma, as reported by others in studies on lymphoid cells (21) and other cancers (24, 25). In contrast to the increased sensitivity to TRAIL induced by LLnL, the latter did not increase the sensitivity of the melanoma lines to apoptosis induced by CD40L, TNF-␣, or FasL, the exception being small increases in sensitivity of one line to FasL. However, proteasome inhibitors are not specific for NF-␬B and are responsible for degradation of proteins involved in cell cycle regulation (41), which might have indirect effects on induction of apoptosis. Therefore, we transfected melanoma cells with a degradation-resistant form of I␬B␣ that binds NF-␬B proteins in the cytoplasm and thereby specifically inhibits activation of NF-␬B. In

Table VII. Inhibition of NF-␬B activation by proteasome inhibitors and by transfection of mutated I␬B␣ increases TRAIL-induced apoptosis of parental and TRAIL-selected resistant melanoma sublines Percentage of TRAIL-Induced Apoptosis

Cell Line

TRAIL Alone

TRAIL ⫹ LLnL

MM200 MM200Sel Mel-RM Mel-RMSel Mel-FH Mel-FHSel

49.0 1.0 24.3 3.4 32 6

86.5 53.7 42.9 55.8 53 62

a b

a

TRAIL ⫹ cells transfected with vector alone

TRAIL ⫹ cells transfected with mutated I␬B␣

48.6 3.8 24.7 8.0 24.2 16.4

74.2 21 50.2 19 52.3 ND

LLnL was used at 2.5 ␮M and added 2 h prior to TRAIL. Apoptosis levels induced by LLnL alone were ⬍6%. Apoptosis levels were ⬍5% in the transfected cells in the absence of TRAIL.


the specificity of these results for inhibition of NF-␬B was obtained by transient transfection of the TRAIL-resistant MM200Sel and Mel-RMSel lines with the degradation-resistant I␬B␣ (66 and 48% transfection efficiency, respectively). As shown in Table VII, the cells transfected with the latter had increased sensitivity to TRAIL-induced apoptosis that was similar to that seen in melanoma cells treated with LLnL. When the transfection efficiency is allowed for, the percentage of apoptosis levels induced by TRAIL approximates that in the lines pretreated with LLnL. The results were similar in a repeat of the study and confirm that activation of NF-␬B was responsible in part for the resistance of the cell lines to TRAIL-induced apoptosis.

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some experiments, cotransfection of a GFP expression vector was used to identify the transfected cells. These studies showed that the transfected melanoma cells had increased sensitivity to TRAILinduced apoptosis and decreased binding of NF-␬B proteins in the nucleus. NF-␬B reporter assays indicated that some melanoma had high constitutive levels of NF-␬B activation but this was not necessarily related to resistance to TRAIL-induced apoptosis. Instead, it appeared from the NF-␬B reporter assays that resistance to apoptosis correlated more with the increase in the level of NF-␬B activation after exposure to TRAIL. This was shown particularly in studies on Mel-FH and Me1007 and in studies on melanoma lines that had been selected for resistance to TRAIL, such as Mel-RMSel. Melanoma cells with high constitutive levels of NF-␬B activation (Mel-JG and MM200) underwent a small increase in NF-␬B activation after exposure to TRAIL and were relatively sensitive to TRAIL-induced apoptosis. High constitutive levels of activated NF-␬B in melanoma were reported previously to be associated with the level of oxidative radicals in the cells (46) or autocrine production of IL-1, which is known to activate NF-␬B (47). A third possible mechanism was accelerated decay of I␬B in melanoma cells allowing NF-␬B proteins to enter the nucleus (48). Reasons for the sensitivity of melanoma cells to TRAIL-induced apoptosis despite the presence of high constitutive levels of activated NF-␬B remain the subject of ongoing studies. Studies on melanoma lines made resistant to TRAIL by prolonged culture in TRAIL indicated that NF-␬B appeared to be activated to a greater extent in the TRAIL-selected resistant lines compared with the parental line. This was consistent with the marked increases in sensitivity to TRAIL-induced apoptosis in the TRAIL-resistant sublines after treatment with LLnL to inhibit NF␬B. Transfection of the degradation-resistant I␬B into the cells also resulted in an increase in TRAIL-induced apoptosis but not to the same level as seen with the proteasome inhibitor. The latter results may reflect the transfection efficiency or indicate that factors other than those involved in activation of NF-␬B are involved in resistance of the cells to TRAIL-induced apoptosis. Some insights into how activation of NF-␬B may inhibit apoptosis resulting from signals triggered from the same receptor was derived from kinetic studies, which suggested that NF-␬B was activated much earlier than the caspase pathway and at lower concentrations of TRAIL; e.g., activation of NF-␬B was evident by 5 min and at concentrations of 1 ng/ml. This compares with reported activation of caspase-8 30 min after exposure to TRAIL (42) and peak activation of the main effector, caspase-3, by 2– 4 h (49) and at concentrations of ⬃100 ng (26). These possible differences in kinetics of the two pathways needs to be confirmed by studies on the induction of NF-␬B proteins involved in inhibition of apoptosis such as the Bcl-2 homologue A1 and members of the IAP family. If confirmed, these findings might suggest that TRAIL may only induce apoptosis when present at high concentrations and that low concentrations may act to increase resistance to apoptosis. In summary, these studies on melanoma lines and TRAIL-selected lines support the view that mechanisms dependent on activation of NF-␬B play a key role in resistance of melanoma cells to TRAIL-induced apoptosis and suggest that inhibitors of NF-␬B may be a valuable adjunct to treatment of patients with TRAIL. Further studies on fresh melanoma tissue and studies in vivo are needed to support these conclusions.


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