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Clinical Cancer Research Vol. 7, 4195-4201, December 2001
© 2001 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Glioma Cells Deficient in Urokinase Plaminogen Activator Receptor Expression Are Susceptible to Tumor Necrosis Factor-{alpha}-related Apoptosis-inducing Ligand-induced Apoptosis1

Bhavani Krishnamoorthy, Bryant Darnay, Bharat Aggarwal, Dzung H. Dinh, Gregory Kouraklis, William C. Olivero, Meena Gujrati and Jasti S. Rao2

Division of Cancer Biology, Departments of Biomedical and Therapeutic Sciences [B. K., J. S. R.], Neurosurgery [D. H. D., W. C. O., J. S. R.], and Neuropathology [M. G.], University of Illinois College of Medicine at Peoria, Illinois 61656; Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [B. D., B. A.]; and Department of Propedeutic Surgery, Athens University School of Medicine, Athens 11526, Greece [G. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The urokinase plasminogen activation system comprises the ligand urokinase plasminogen activator and the receptor urokinase plasminogen activator receptor (uPAR), which play an important role in the activation of matrix-degrading enzymes that enhance the invasion of cancer cells. Earlier studies have indicated that SNB19 glioblastoma cells expressing antisense uPAR constructs lose their invasive properties when injected in vivo. Additional observations indicated that injected antisense uPAR:SNB19 cells were being lost through apoptotic elimination.

Experimental Design: SNB19, Vector, and SNB19:asuPAR were analyzed to determine cytotoxicity of tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL), receptor expression, and underlying signaling pathways using flow cytometry, immunohistochemistry, RNase protection assay, and c-Jun-NH2-terminal kinase activity.

Results: This study elucidated the susceptibility of antisense uPAR:SNB19 cells to TRAIL under certain experimental conditions in vitro. These uPAR-deficient transfected cells had higher levels of the TRAIL receptors DR4 and DR5 than did the control and vector population as detected by flow cytometry. An RNase protection assay confirmed the elevation of DR4 and DR5 mRNA in the antisense uPAR cells.

Conclusions: These findings provide preliminary evidence of a link between TRAIL-induced apoptosis and cell cycle progression in antisense uPAR:SNB 19 cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interactions between urokinase plasminogen activator and uPAR3 play a role in tumor invasion and metastasis (1 , 2) by the activation of matrix-degrading proteases. A direct correlation has been established between the expression of uPAR and invasion in glioma cells, where introduction of an antisense construct of the uPAR gene into SNB19 glioma cells rendered the transfectants noninvasive (3, 4, 5) and apoptotic (6) . uPAR also has additional roles as a growth factor receptor or transducing agent, e.g., uPAR signaling pathways seem to share common signaling complexes with the mitogenic signaling pathway, and some autocrine or paracrine functions may require interaction with surface uPAR for the generation of growth stimuli. uPAR-deficient cells have been shown to enter a state of dormancy, surviving without progressive growth for <=5 months. Reduced expression of this receptor may diminish the responses that are mediated via this receptor, such as migration, mitogenicity, or induction of neovascularization (7) .

In one study, down-regulation of uPAR in human carcinoma HEp3 cells led to arrest of the cells in G0-G1 in vivo but did not affect growth in vitro (7) . Aguirre Ghiso et al. (8) attributed this dormancy to a reduced expression of {alpha}5ß1 receptor in these cells that led to a lesser adhesion to fibronectin. Basal levels of extracellular signal-regulated kinase1/2 were reduced by four to six times in uPAR-poor cells, perhaps because of lower levels of interaction with the fibronectin receptor. Binding of urokinase plasminogen activator to uPAR has been shown to signal the coclustering of casein kinase-2, a serine threonine kinase involved in mitogenic signaling, and nucleolin, a ribonucleoprotein that shuttles between the nucleus and the cytosol (9) . Although no evidence has been found to suggest a direct correlation between suppression of uPAR expression and apoptosis in the malignant phenotype, these observations imply that down-regulation of uPAR and its associated signals may render a cell vulnerable to apoptotic stimuli.

TRAIL and its receptors DR4 and DR5 are part of a powerful apoptotic process that is tightly regulated. The TRAIL family of receptors comprises four members: TRAIL-R1 (DR4, Apo-2), TRAIL-R2 (DR5), and the two decoy receptors DcR1 (TRAIL-R3, TRID, and LIT) and DcR2 (TRAIL-R4). The structure of the TRAIL receptors DR4 and DR5 is similar to those of the TNF superfamily, consisting of a ligand-binding extracellular domain, a transmembrane anchor, and the intracellular death domain Fas-associated death domain, which is associated with caspases-8 and -10. The decoy receptors differ in that they either lack the intracellular tail (DcR1) or have a truncated death domain (DcR2). Both decoy receptors are expressed on normal cells and can harness TRAIL, prevent cytotoxic signaling, and render the cell resistant to TRAIL, whereas the DR4 and DR5 receptors are abundant on cancer cells (10) . Several cell lines exhibit resistance to induction of apoptosis mediated by ligand-receptor interactions that can be overcome by treatment with cycloheximide or actinomycin D, suggesting that these cells produce one or more resistance factors by active transcription/translation (11) .

TRAIL has demonstrated apoptosis induction in epithelial-derived cancers (12) , melanomas (13) , and breast cancer (14) . A synergistic effect has been observed in the mode of induction of apoptosis by TRAIL with genotoxic agents and radiation, suggesting that the combination of chemotherapy (15) or radiation (11 , 14 , 16 , 17) with TRAIL may be an effective treatment for many cancers. The signaling pathways underlying the association of TRAIL with its receptors are well delineated (10 , 18) , but increasing evidence seems to suggest that other signals that govern cell cycle progression also seem to converge on this pathway, influencing the susceptibility to TRAIL. Cell cycling and apoptosis are governed by common gatekeepers in signaling mechanisms (19 , 20) , and often, the induction of apoptosis can be brought about by a slip in the mitogenic signaling pathway. The expression of TRAIL receptors DR4 and DR5 has been shown to be influenced by expression of cyclin D. In cells overexpressing cyclin D, etoposide induces an increase in the expression of DR4 and DR5, suggesting a possible therapeutic strategy to combat breast cancer (14) .

uPAR is a glycosyl-phosphatidylinositol-anchored protein that is localized to invaginations of the plasma membrane called caveolae, which create the ideal microenvironment to promote paracrine interactions. uPAR indulges in cross-talk with other signaling complexes, such as the {alpha}5ß1 integrins and the Ras-Raf-1-mitogen-activated protein/extracellular signal-regulated kinase kinase pathway (21 , 22) . Despite the lack of a transmembrane anchor, uPAR efficiently transduces signals through a gamut of Src tyrosine kinases, as well as ß1, ß2, and ß3 integrins (23) . The death receptor TNF-R1 and decoy receptor DcR1 are also glycosyl-phosphatidylinositol-anchored caveolar proteins (24) . Thus, it is reasonable to assume that the close proximity of these receptors and, therefore, their ligands may encourage cross-talk between their respective signaling pathways.

The findings described in this paper suggest a novel pathway linking urokinase-receptor signaling and TRAIL-mediated apoptosis. SNB19 glioblastoma cells lacking the urokinase receptor showed increased susceptibility to TRAIL when de novo protein synthesis was inhibited with cycloheximide. SNB19 cells expressing the antisense uPAR constructs also possessed greater levels of DR4 and DR5 than did the parental cells. This latter finding probably reflected the increased cytotoxicity of TRAIL, but what emerges from these studies is that the mere overexpression of the death receptors was not sufficient to increase cytotoxicity to TRAIL. Rather, cell death ensued only on treatment with cycloheximide, implying that a cytoprotective mechanism exists in the antisense clones that was inhibited by protein synthesis inhibition.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents.
Recombinant human TRAIL and anti-DR4 and -DR5 antibodies were obtained from Imgenex (San Diego, CA). Propidium iodide, MTT, protein A-Sepharose, and other chemicals were obtained from Sigma Chemical Co. SNB 19 cells were obtained from American Type Culture Collection, and the antisense clones were developed as described earlier (3 , 4) . Cells were cultured in DMEM/F12 High-Glucose medium with 10% FCS (Life Technologies, Inc.). Recombinant GST-Jun was produced in the laboratory according to a standardized protocol (25) . Multiprobe templates for the RNase protection assay were obtained from PharMingen, and the RPAIII kit was obtained from Ambion, Inc.

Evaluating Cytotoxicity of TRAIL in SNB19 Glioma Cells by MTT Assay.
SNB19 glioma cells were seeded in 96-well plates (5 x 105 cells/well) and analyzed for TRAIL-induced cytotoxicity with the MTT assay. Glioma cells were treated with TRAIL (20 ng/ml), cycloheximide (1 µg/ml), or TRAIL (20 ng/ml) plus cycloheximide (1 µg/ml). Cells were incubated for 24 h at 37°C in a 5% CO2 incubator. Medium was then aspirated, and cells were stained with MTT solution (1 mg/ml) at 37°C for 3 h, after which, the dye was extracted by the addition of dimethylsulfoxide, and absorbance was read at 550 nm.

Detection of Apoptosis in Glioma Cells by Propidium Iodide Staining and Flow-cytometric Analysis.
Cells (1 x 106) were trypsinized and washed in ice-cold PBS with 1% BSA three times and incubated with 100 µg/ml RNase A for 30 min at 37°C. Propidium iodide (50 µg/ml) was added, and cells were stained for 20 min at 4°C or left in this solution until FACS analysis with an EPICS Elite flow cytometer (Coulter).

Identification of DR4 and DR5 Expression by Flow Cytometry and the RNase Protection Assay.
SNB19:AS-uPAR cells were stained for DR4 and DR5 by using receptor NH2-terminal-specific antibodies that were detected with FITC-tagged secondary antibodies. Cells (1 x 106) were analyzed with an EPICS Elite flow cytometer. Fluorescence data from the vector- and antisense-transfected cells were overlaid with those of the parental SNB19 cells.

The mRNA expression levels of the TNF-receptor superfamily and related genes and cell cycle regulators were compared among parental, vector-control, and antisense-transfected cells with the RNase protection assay using hApo-3d and hStress-1 probe templates (PharMingen). Probe transcription was carried out by using {alpha}-32P UTP (800 Ci/mmol) in transcription mix (Ambion) containing 1 x transcription buffer and 0.5 mM ATP, CTP, and GTP and T7 RNA polymerase and RNase inhibitor. This was followed by DNase treatment and phenol:chloroform isoamylalcohol extraction, and the transcripts were resuspended in hybridization solution. RNA (10 µg) was hybridized with either hApo-3d (4.5 x 105 cpm/µl) or hStress-1 (2.9 x 105 cpm/µl) probe overnight at 56°C. The tubes were treated with RNase A/T1 for 1 h at 30°C, inactivated, precipitated, dried, and then resuspended in loading buffer. Yeast RNA- and RNaseA/T1-negative controls were maintained. Samples were loaded onto 40-cm 5% acrylamide/7 M urea sequencing gels and run at 20–25 V/cm. The gels were then dried and exposed to autoradiographic film.

JNK Activity Assay.
Cells were lysed in lysis buffer [20 mM Tris (pH 7.4), 250 mM NaCl, 0.2% Triton X-100, 1 mM DTT, 1 mM sodium orthovanadate, 2 µg/ml aprotinin and leupeptin, and 1 mM EDTA] for 30 min on ice. Lysates were centrifuged at 14,000 rpm for 10 min at 4°C, after which, supernatants were collected, and protein was estimated. Protein (40 µg) was then immunoprecipitated with anti-JNK antibody (Santa Cruz Biotechnology) for 1 h at 4°C after which, protein A-Sepharose (50% slurry in PBS) was added. The tubes were rocked end-over-end for 30 min at 4°C and then centrifuged at 10,000 rpm for 5 min at 4°C. The pellets were washed carefully three times in lysis buffer, with care taken to aspirate the washes completely. Pellets were then washed two times in low-salt buffer [25-mm HEPES (pH 7.9), 25 mM NaCl, and 1 mM DTT], resuspended in 10 µl of reaction buffer [10 mM MgCl2, 25 mM HEPES (pH 7.9), 1 mM DTT, 2 µg of GST-Jun, and 10 µCi of {gamma}-32P ATP], and incubated at 30°C for 10–30 min. Then, 15 ml of 2 x sample buffer were added to the reaction mixture, and the samples were heated to 95°C for 5 min and loaded on a 10% SDS-PAGE gel. The gel was stained with Coomassie Blue to visualize the amount of total protein, exposed to autoradiographic film or phosphor screen, and analyzed with a Molecular Dynamics PhosphorImager (Molecular Dynamics, Hayward, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Estimation of Cytotoxicity of TRAIL on Glioma Cells by MTT Assay.
Fig. 1ACitation shows that both parental SNB19 and vector control cells are resistant to treatment with TRAIL in combination with cycloheximide. However, the viability of antisense uPAR cells undergoes a significant reduction (P < 0.001) on cotreatment with TRAIL and cycloheximide. This preliminary result prompted the investigation of the mode of cell death in antisense uPAR cells on exposure to cycloheximide and TRAIL.



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  		Fig. 1. A, cytotoxicity of TRAIL and cycloheximide using the MTT assay. Cells were treated with TRAIL, cycloheximide, or both for 24 h. The graph representing the mean of three separate experiments indicates that either TRAIL or cycloheximide was nontoxic to the parental, vector control, and SNB19:antisense uPAR cells, but treatment with both TRAIL and cycloheximide decreased the viability of the antisense clones (* denotes P < 0.001). B, percentage of apoptotic cells. Cells were stained with propidium iodide after treatment with TRAIL, cycloheximide, or both and subjected to FACS analysis of apoptosis. Treatment of the antisense uPAR population with both TRAIL and cycloheximide produced a shift toward sub-G0. Controls for untreated cells and cycloheximide-treated cells indicated normal cycling.

 
Detection of Apoptosis in Glioma Cells by Propidium Iodide Staining and FACS Analysis.
SNB19 cells were tested for susceptibility to apoptosis by analyzing DNA content with propidium iodide staining followed by FACS analysis. Parental SNB cells and vector controls are relatively stable cell lines; their cycling is not affected by treatment with cycloheximide (1 µg/ml) or TRAIL (20 ng/ml for 24 h). However, the antisense uPAR-transfected SNB19 cell line is inherently unstable and undergoes excessive cell death on serum deprivation.4 Untreated control antisense uPAR cells showed a basal level of apoptosis (6%) that increased marginally on treatment with cycloheximide (11.8%). The addition of TRAIL to cycloheximide-treated cells increased the percentage of apoptosis substantially (30%). However, the entire population of cells was observed to be floating and had apoptotic morphology. FACS analysis also clearly indicated a consistent shift in cell-cycle distribution toward sub-G0 in the antisense uPAR cells treated with TRAIL and cycloheximide (Fig. 1B)Citation . These findings show that TRAIL induced apoptosis in antisense uPAR cells cotreated with cycloheximide.

DR4 and DR5 Expression by Flow Cytometry and RNase Protection Assay.
Expression of the death receptors was confirmed at the protein level by immunocytochemistry followed by flow-cytometric quantification and at the mRNA level by the RNase protection assay, which uses multiprobe templates to evaluate expression of signaling mediators of the death receptor pathway. Flow-cytometric analysis of DR4 and DR5 expression revealed that the antisense uPAR clones of SNB19 glioma cells expressed 97.2 and 99.5% more surface receptors than did the control parental cells and vector cells, respectively (Fig. 2)Citation . Comparison of the mRNA expression levels of TNF-receptor superfamily and related genes with the RNase protection assay indicated complete silence in the deathreceptor also known as CD95/death receptor ligand pathway in SNB19 cells. Antisense uPAR cells showed elevated levels of caspase-8, DR4, and DR5 relative to those in parental and vector SNB19 cells (Fig. 3)Citation . There was no significant difference in expression levels of Decoy Receptors (DcR1 and DcR2) by any of the above methods of detection.



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  		Fig. 2. Flow-cytometric determination of DR4 and DR5 expression. Cells were stained with antibodies against the NH2 terminus of DR4 and DR5 and tagged with FITC-conjugated secondary antibodies. Receptors were present at higher levels in the SNB19:as-uPAR cells than in the parental cells.

 


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  		Fig. 3. RNase protection assay using a multiprobe template for the TRAIL receptor signaling pathway. Lane P, unprotected probe; Lane R-, RNase-treated probe control; Lane S, SNB19 cell lysate; Lane V, vector cell lysate; Lane As, SNB19:as-uPAR cell lysate. Expression of caspase-8, DR4, and DR5 mRNA in antisense uPAR cells was elevated over that in parental and vector cells.

 
The RNase protection analysis was also done with the hStress-1 multiple-probe cell cycle-regulator template (PharMingen). This assay revealed a down-regulation of expression of Bcl-2 family members in the antisense clones relative to that in the parental and vectors cells. Specifically, the antisense uPAR:SNB19 cells expressed half as much Bcl-xL and mcl-1 and much less Bax and Bcl-2 than did the parental SNB19 cells. Comparison of the SNB19 and SNB19:AS-uPAR cell lines also showed no p21 expression in the antisense cells (Fig. 4)Citation . These results support the hypothesis that a cell cycle aberration may be responsible for the TRAIL-induced cytotoxicity in antisense uPAR glioma cells.



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  		Fig. 4. RNase protection assay using a multiprobe template for cell cycle regulators and apoptotic markers. Cycloheximide (cyc) was used at 1 µg/ml, and TRAIL was used at 20 ng/ml. Lane P, unprotected probe; Lane R-, RNase-treated probe control; Lane 1, SNB19 + cyc; Lane 2, SNB19 + cyc + TRAIL; Lane 3, SNB19:AS-uPAR + cyc; Lane 4, SNB19:as-uPAR + cyc + TRAIL. Expression of mRNA for Bcl-xL and mcl-1 was down-regulated, and the expression of mRNA for bcl-2 and bax was completely silenced in antisense uPAR cells on treatment with cycloheximide and TRAIL. The vector mRNA profile (data not shown) was identical to that of the parental cells.

 
JNK Activity Assay.
Lysates of glioma cells exposed to either cycloheximide (1 µg/ml) or cycloheximide (1 µg/ml) and TRAIL (20 ng/ml) were analyzed for levels of JNK activity by using {gamma}-32P-labeled GST-Jun substrate. The SNB19 parental and vector cells behaved identically with respect to JNK activation, i.e., these cells showed a basal level of JNK activity that was down-regulated on treatment with either cycloheximide alone or cycloheximide plus TRAIL. However, antisense uPAR cells displayed similar levels of JNK activity in the untreated and cycloheximide-treated conditions but showed a decrease in JNK activity only in the cotreated condition. There was no change in JNK activity on the addition of TRAIL alone in any of these cell lines, confirming that the effect observed occurred only in combination with cycloheximide (Fig. 5)Citation .



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  		Fig. 5. JNK activation in cell lysates using GST-Jun-specific substrate phosphorylation with {gamma}32P ATP. Parental SNB19, vector, and antisense uPAR cells were treated as indicated, and 40 µg of the lysates were used for the activity assay. Treatment with cycloheximide inhibited JNK activity in parental and vector cells but not in antisense uPAR cells. The latter underwent partial JNK inhibition on cotreatment with TRAIL.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TRAIL has been heralded as a panacea in cancer therapy because of its ability to specifically target transformed cells and leave healthy cells unaffected. Several types of cancer have been shown to be susceptible to TRAIL-induced cytotoxicity, including melanoma (13) , breast carcinoma (14) , and glioblastoma (26) . However, little is known about the mode of action of TRAIL’s lethal effect or its apparent inconsistency in inducing apoptosis in some cell types. Recent evidence suggests a connection between TRAIL-induced apoptosis and cell cycle progression (14 , 26 , 27) , indicating that TRAIL exerts a cytotoxic effect only if the integrity of the cell cycle is compromised.

TNF-receptor-mediated signaling requires that de novo protein synthesis be blocked with cycloheximide to induce apoptosis, but TRAIL can induce apoptosis of H4 glioma cells even in the absence of this protein synthesis inhibitor.5 In the present study, SNB19, an aggressive glioblastoma cell line, was resistant to TRAIL-induced apoptosis in the presence or absence of cycloheximide (Fig. 1A)Citation . The introduction of an antisense construct to uPAR, an invasion-promoting enzyme, renders SNB19 cells susceptible to TRAIL, perhaps because: (a) the silencing of uPAR expression with the antisense construct somehow blocks mitogenic signals from feeding into the cell cycle, thus destabilizing it; or (b) treatment with cycloheximide inhibits production of cell cycle regulatory proteins, leading to cell cycle stress. Additional investigations of cell cycle regulators will provide insight into the pathways involved.

The role of the Bcl-2 family of proteins in fine-tuning the apoptotic cascade is well understood (28) . In the present study, we observed a general down-regulation of all Bcl-2 family members during TRAIL-mediated apoptosis (Fig. 4)Citation , including a reduction in Bax (proapoptotic function), as well as Bcl-xL, Bcl-2, and mcl-1 (antiapoptotic function). Although bcl-2 is known to rescue cells from a wide range of apoptotic stimuli (28) , some debate persists about its ability to promote survival in death receptor-mediated apoptosis (29 , 30) . Future investigations will clarify the role of these antiapoptotic mediators and cell cycle regulators in TRAIL-induced apoptosis.

p21WAF1/CIP1 plays an important role in the p53 signaling pathway and in G1 arrest. Recent findings showed an association between levels of p21 and TRAIL-induced apoptosis (31) . Wu et al. (26) suggested that the DR5 receptor is under the transcriptional control of p53 and observed that treating p53-expressing cells with actinomycin D reduced p21 and bax expression. We saw a decline in p21 expression in the antisense clones relative to that in the parental cells (Fig. 4)Citation . The absence of a major participant in the cell cycle may be one reason why antisense clones are easy targets of cytotoxic agents, such as TRAIL. This finding also directs attention toward a possible association between TRAIL susceptibility and cell cycle destabilization.

The JNK is an active signaling modulator in cell stress pathways in response to environmental genotoxic stimuli, as well as mitogenic stimuli. The role of JNK kinases in apoptotic signaling has only recently been recognized, and their effects seem to be specific to cell types (32) . Although JNK has been shown to be activated by ligation of death receptors (33) , the exact role of JNK in death receptor-mediated apoptosis is unclear (32) . TNF-{alpha} is known to activate JNK pathways during apoptosis, but JNK activation and apoptosis can be dissociated by using different components of the TNF-{alpha} receptor complex (18 , 34) . Our results here showed that treatment of SNB19 parental and vector control cells with cycloheximide led to a drop in JNK activity below basal levels (Fig. 5)Citation , suggesting that de novo protein synthesis is a prerequisite for JNK activation. Cotreatment with TRAIL did not enhance JNK activity in these cells. However, the antisense uPAR clones showed no reduction in JNK activity levels on treatment with cycloheximide, suggesting that the inhibition of JNK activity seen in the parental and vector cells was somehow overcome. Treating the antisense clones with TRAIL and cycloheximide, however, did down-regulate JNK activity. These observations indicate that the antisense uPAR clones were unaffected by blockage of de novo protein synthesis and may possess a mechanism to elude this requirement.

These findings lead us to believe that the JNK pathway is neither necessary nor a sufficient executioner of the apoptotic pathway, but rather, it is one of the pathways activated or inhibited during the apoptotic cascade that leads to cellular dysfunction. The exact role of the Bcl-2 family in this sequence of events has yet to be further investigated. This work is the first evidence of the existence of overlapping pathways between uPAR signaling and the death receptors of the TRAIL pathway. This preliminary evidence points toward a convergence of these two pathways at the level of the cell cycle, where the disruption of uPAR signaling offsets the progression of cell cycle, thus rendering the cell susceptible to apoptotic stimuli, such as TRAIL. Additional studies are under way to identify the locus of convergence locus of these two pathways.


   ACKNOWLEDGMENTS
 
We thank Dr. Ram Reddy and Karthika Perumal, Baylor College of Medicine, Houston, Texas, for their assistance with the RNase protection assay. We also thank Karen M. Ramirez at the Flow Cytometry Core Facility of The University of Texas M. D. Anderson Cancer Center for her help with FACS analysis.


   FOOTNOTES
 
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.

1 Supported by National Cancer Institute Grant CA 75557 (to J. S. R.) Back

2 To whom requests for reprints should be addressed, at Division of Cancer Biology, Departments of Biomedical and Therapeutic Sciences and Neurosurgery, University of Illinois, Peoria, IL 61656. Back

3 The abbreviations used are: uPAR, urokinase plaminogen activator receptor; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-{alpha}-related apoptosis-inducing ligand; JNK, c-Jun-NH2-terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorting. Back

4 , unpublished observations. Back

5 B. Aggarwal and B. Darnay, manuscript in preparation. Back

Received 5/ 8/01; revised 8/ 9/01; accepted 8/31/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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D. Alfano, I. Iaccarino, and M. P. Stoppelli
Urokinase Signaling through Its Receptor Protects against Anoikis by Increasing BCL-xL Expression Levels
J. Biol. Chem., June 30, 2006; 281(26): 17758 - 17767.
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