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Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 [M. J. F., R. A.]; Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 [A. K. V., A. F. G.]; Department of Head and Neck Surgery, The Johns Hopkins University, Baltimore, Maryland 21205 [L. W., D. S.]; Division of Gastroenterology and Hepatology, The University of Virginia Health System, Charlottesville, Virginia 22906 [J. C. H., S. M. P.]; Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 [T. R. R.]; and Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute [M. J. F., W. S. E-D.], and Departments of Medicine, Pharmacology, and Genetics [W. S. E-D.], University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 = 5.2, P = 0.023). The frequency of homozygosity for both alleles was also significantly increased in the primary NSCLC samples (2 = 9.2, P = 0.002) as compared with the age- and race-matched controls. To determine whether the altered alleles are specific for lung cancer, we evaluated 19 head and neck squamous cell cancer and 25 gastric adenocarcinoma samples. Forty-seven % of the former and 44% of the latter were homozygous for both the C626G and G422A alterations, and this was significantly elevated relative to age- and race-matched controls (2 = 8.6, P = 0.003 and 2 = 8.2, P = 0.004). These alterations result in amino acid changes in or near the ligand-binding domain of DR4 and, based on the crystal structure of DR5 and its homology with DR4, have the potential to affect TRAIL binding to DR4. Our results suggest that the altered DR4 alleles may be associated with, and should be investigated additionally as potential markers for, predisposition to common malignancies. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiple genetic lesions have been described in lung cancer (2) . Mutations of both dominant oncogenes and of tumor suppressor genes have been noted. Allelic loss (LOH) at chromosomal regions 3p and 9p is noted in both invasive lung cancer and preneoplastic lesions (2, 3, 4) . Allelic loss of chromosome 8p21–22 is a frequent event in various cancers including lung, prostate, colon, hepatocellular carcinoma, and HNSCC (5, 6, 7, 8, 9) . Deletions of 8p21–23 have been detected in high frequency as an early event in both SCLC and NSCLC cell lines and primary tumors (10) .
The family of TRAIL receptors, including the proapoptotic DR4 and KILLER/DR5, as well as the decoy receptors TRID and TRUNDD, are all located on human chromosome 8p21–22 (11, 12, 13, 14) . DR4 and KILLER/DR5 have significant homology with the other members of the tumor necrosis factor receptor family, which are characterized by a cysteine-rich, extracellular ligand-binding domain (14) . Signaling through DR4 and KILLER/DR5 is dependent on the presence of a cytoplasmic DD in these receptors (15) . Binding of TRAIL to DR4 and KILLER/DR5 initiates a signaling pathway that triggers a caspase cascade, which results in apoptosis (15 , 16) . TRAIL receptors are excellent candidate tumor suppressor genes, because their inactivation would be expected to result in deficient apoptotic signaling.
Mutations in the DD of KILLER/DR5 have been noted in both HNSCC (17) and lung cancer (18) . In the former case, the mutation resulted in the loss of the proapoptotic function of the receptor. To date, no reports of DR4 mutations in human cancer have been identified. Previous work has identified a polymorphism in the DD of DR4 in both a bladder and ovarian cell line and in 20% of normal individuals (19) . This polymorphism was an A-to-G transition at nucleotide 1322 (A1322G), resulting in the conversion of amino acid lysine (codon 441) to arginine (K441R). To additionally investigate the involvement of DR4 in human cancer, we screened 31 lung cancer cell lines (14 SCLC and 17 NSCLC), many with deletions at 8p21–23, and 21 primary NSCLC specimens for mutations in DR4. We first characterized DR4 by defining its exon-intron boundaries and generating a detailed physical map of the coding region. We then evaluated for DR4 mutations by direct sequencing of genomic DNA derived from tumor and normal samples. The results reveal two alterations in the ectodomain of DR4 that occur together in a homozygous fashion at an increased frequency in lung cancer cell lines (P = 0.023) and primary NSCLC (P = 0.002), HNSCC (P = 0.003), and gastric adenocarcinoma (P = 0.004) samples relative to age- and race-matched controls. These results suggest that the altered DR4 alleles may be associated with a predisposition to common malignancies.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor and Matched Normal Specimens.
Twenty-one paired primary NSCLC samples and their corresponding matched DNA from adjacent, normal lung tissue were analyzed. DNA was isolated as described previously (20)
. This group was 100% Caucasian with a median age of 66 years (range, 37–87 years) and consisted of 65% males and 35% females (demographic data were available on all but 1 of the specimens). Nineteen primary HNSCC samples were evaluated. DNA was derived from freshly frozen tissue as described previously (17)
. Complete demographic data were available for all but 2 of the specimens. The median age of this group was 62 years (range, 41–86 years) with 76% males, 24% females, 88% Caucasians, and 12% African-Americans. No matched, normal DNA was available for these samples. Twenty-five surgically resected, gastric adenocarcinoma specimens were collected in standard RPMI 1640 and stored on ice for xenografting. Normal tissue from resected specimens or peripheral blood samples was also procured. Xenografting was performed as described previously (21)
. Briefly, small pieces of tumor tissue were soaked in Matrigel (Collaborative Biomed Research) and then implanted s.c. into the flanks of immunodeficient mice (nu/nu mice from Harlan or severe combined immunodeficient mice from Charles River) for xenografting growth. Passage tumors were harvested when their diameter reached 
1 cm, and genomic DNA was then extracted by a standard organic method as described previously (22)
. Corresponding normal DNA from each patient was also extracted in a similar fashion. Complete demographic data were available on only 16 of these specimens. This group was 100% Caucasian with a median age of 65 years (range, 41–83 years) and composed of 62% males and 38% females.
Normal Controls.
Three groups of normal volunteers were evaluated. Group 1 consisted of 10 individuals (70% male, 30% female; 50% Caucasian, 50% Asian; median age, 36 years; age range, 25–43 years) and has been described previously (19)
. Group 2 consisted of 15 individuals. DNA was isolated from peripheral blood lymphocytes (20)
after centrifugation over a ficoll gradient. This group had a median age of 34 years (range, 26–57 years) and was 60% male, 40% female, 60% Asian, and 40% Caucasian. Group 3 consisted of 45 individuals frequency matched for age and race to the combined lung cancer cell line, primary NSCLC, and HNSCC groups. For each individual group comparison, there were no statistically significant differences in race between the cases and the controls (P > 0.5 in each case). DNA was obtained and processed from the group 3 individuals as described previously (23)
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Exon-Intron Boundaries.
Exon-intron boundaries were determined by direct DNA sequencing of subclones derived from EcoRI digestion of a P1 clone containing the DR4 gene (Genome Systems clone 18847). Template DNA was isolated by CsCl banding. Sequencing was performed using the T7 Sequenase Version 2.0 DNA Sequencing kit (United States Biochemical Corp.) as per the manufacturer’s instructions. Primers were designed from the known cDNA sequence. After sequencing, samples were heated to 75°C for 2 min and loaded on a 6% denaturing sequencing gel. Primers used in sequencing to determine exon-intron boundaries were as follows: DR4-7, 5'-AAGCCAGCCCTCGGCTCCGGGT-3'; DR4-3R, 5'-GGACACAACTCTCCCAAAG-3'; DR4-8, 5'-CAGGTCGTACCTAGCTCAGC-3'; DR4-2, 5'-CATCTGATTTACAAGCTGTACA-3'; DR4-23, 5'-AACGTCCTGGAGCCTGTAACCG-3'; DR4-20, 5'-TGTGCTGCACTTCCGGCACATC-3'; DR4-24, 5'-AGAGAAGTCCCTGCACCACG-3'; DR4-5R, 5'-AACCAAAATCACCCATATATTAT-3'; DR4-21, 5'-ACAACAGACAATCAGCACAG-3'; DR4-17, 5'-GCAATGGACATAATATATGG-3'; DR4-15S, 5'-AACACTGCCCTCTAAGAAGG-3'; DR4-16, 5'-AGCAGACACTGTGCCTCCCC-3'; DR4-19, 5'-TGGCGCTTGGGTCTCCTACG-3'; DR4-10, 5'-TCTCAGTGGGGTCAGCACCA-3'; DR4-18, 5'-GACCGGCAGAAGCTGAAGGG-3'; and DR4DD-2R, 5'-CTTCTCTTTTGCATGTCTCTCTTCC-3'.
Genomic Map of the Human DR4 Coding Region.
Most intron sizes were determined by PCR amplification of each intron using the DR4 P1 clone as a template. Primers were designed from the known cDNA sequence as well as intronic sequence derived during the course of sequencing for exon-intron boundaries. The primer pairs for each intron were as follows: intron 3, DR4-23 and DR4-20; intron 4, DR4-24 and DR4-I4R (5'-TTCTTTGTGGACACACTCG-3'); intron 6, DR4-17 and DR4-15 (5'-ACTTGGGGTCCCCTCCACAA-3'); intron 7, DR4-22 (5'-TGTGGAGGGGACCCCAAGTG-3') and DR4-13 (5'-CCCTCGTAGGAGACCCAAGC-3'); intron 8, DR4-19 and DR4-10; and intron 9, DR4-18 and DR4DD-2R. PCR amplification was carried out in 50-µl reactions consisting of 1x buffer [16.6 mM (NH4)2SO4, 67 mM Tris-HCl (pH 8.8), 6.7 mM MgCl2, and 10 mM ß-mercaptoethanol], 0.35 mM each deoxynucleoside triphosphate (dATP, dTTP, dCTP, and dGTP), 150 ng of each primer for a given intron as above, 5 units of Taq DNA polymerase (Perkin-Elmer Biosystems), and 100 ng of DR4 P1 clone template DNA. PCR conditions consisted of an initial denaturation step of 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C–68°C (depending on the primer pair) for 1 min, extension at 72°C for 2 min, and a final elongation step of 72°C for 5 min. The PCR products were electrophoresed on a 1% agarose gel, and band sizes were determined. Intron 5 size was determined by direct sequencing of the P1 clone as described above using primer DR4-21. Intron 2 size was determined using nested PCR and direct sequencing. Initial PCR was performed using primers DR4-I2F (5'-CAATTGGCACACAGCAATGG-3') and DR4-I2RS (5'-GGGAGGCAAGCAAACAAATTG-3'). One µl of the 50-µl PCR product was used in a second PCR reaction with primers DR4-3 (5'-CTTTGGGAGAGTTGTGTCC-3') and DR4-I2R (5'-AGGACGTTCTGATCTATGAG-3'). PCR conditions for both reactions were as follows: initial denaturation step of 94°C for 3 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 45 s, extension at 72°C for 2 min, and a final elongation step of 72°C for 5 min. After electrophoresis on a 2% agarose gel to determine band size, the band was purified using a QIAquick Gel Extraction kit (Qiagen) as per the manufacturer’s instructions. The product was sequenced using the nested set of primers (DR4-3 and DR4-I2R) to confirm both intron-exon boundaries. Sequencing was performed using the SequiTherm Cycle Sequencing kit (Epicentre Technologies). After performing the sequencing reaction, samples were heated to 80°C for 5 min, loaded on a 6% denaturing polyacrylamide gel, and processed as described previously (19)
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Genomic Sequencing.
Sequence analysis of the DD in exon 10 as well as the cysteine-rich, extracellular ligand-binding domain coded for by exons 3 and 4 of DR4 was performed using all of the 31 lung cancer cell lines and their matched B-lymphoblastoid cell lines. Each region was amplified from genomic DNA by PCR. Primers pairs were as follows: DD, DR4-11 (5'-CTCTGATGCTGTTCTTTGAC-3') and DR4-12 (5'-TCACTCCAAGGACACGGCAG-3'); exon 3, DR4-I2 (5'-ATCCTCTGGGAACTCTGTGG-3') and DR4-E3R (5'-TACCACTCCCACCTCACTGC-3'); and exon 4, DR4-E4F (5'-AAGGTCAAGGGACACGTCAGG-3') and DR4-E4R (5'-GCTTCTGTGGTTTCTTTGAGG-3'). PCR amplification was performed in 50-µl reactions consisting of 1x PFU buffer (Stratagene), 0.35 mM each deoxynucleoside triphosphate (dATP, dTTP, dCTP, and dGTP), 150 ng of each primer for a given exon as listed above, 2.5 units PFU Turbo DNA polymerase (Stratagene), and 50 ng of template DNA. PCR conditions were as follows: initial denaturation step of 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 45 s, extension at 72°C for 1 min, and a final elongation step of 72°C for 5 min. After electrophoresis on a 2% agarose gel, the bands were purified using a QIAquick Gel Extraction kit (Qiagen) as per the manufacturer’s instructions. Sequencing was performed using the forward primer and the SequiTherm Cycle Sequencing kit (Epicentre Technologies) as described above.
RFLP Analysis of C626G and G422A Alleles.
To detect the C626 and 626G alleles, exon 4 was amplified as described above. Thirty-nine µl of the PCR product was then incubated in a 50-µl reaction with 20 units DraIII, 0.1 mg/ml BSA, and 1x NEB buffer #3 (New England BioLabs, Inc.) at 37°C for 90 min. The products were then separated on a 2% agarose gel. To detect the G422 and 422A alleles, exon 3 was amplified as described above. Thirty-eight µl of the PCR product was then incubated in a 50-µl reaction with 8 units of FokI, 0.1 mg/ml BSA, and 1x NEB buffer #4 (New England BioLabs, Inc.) at 37°C for 90 min. The products were then separated on a 2% agarose gel.
Statistical Analysis.
Statistical analysis was performed using the STATA software program. Unless otherwise specified, comparisons were made between each tumor group and the age- and race-matched control group.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. There are 10 exons and 9 introns in the coding region. Exon sizes vary from 32 to 320 bp, and intron sizes vary from 86 to 4700 bp. Exact exon-intron boundaries were determined by consensus splice site rules with all of the introns beginning with the dinucleotide GT and ending with AG. Table 1
shows the exon-intron boundaries and the exon sizes. Of note, the genomic structure is strikingly similar to the genomic structure of DR5, both with 10 exons, 9 introns, and similar intron and exon sizes (17)
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Identification of a C626G Allele in Exon 4 of TRAIL Receptor DR4 in Lung Cancer.
Binding of TRAIL to its proapoptotic receptors, KILLER/DR5 and DR4, induces apoptosis (15)
. The ligand-binding motif of the receptors is a cysteine-rich domain, which is characteristic of the extracellular domain of the tumor necrosis factor receptor family. The principle elements of this domain are encoded by exons 3 and 4 of DR4. We performed sequence analysis of exons 3 and 4 in 31 lung cancer cell lines. Cycle sequencing of exon 4 revealed that 21 (68%) of the lung cancer cell lines had a missense alteration at nucleotide 626 changing a cytosine to a guanine (C626G), resulting in a substitution of an arginine for threonine at codon 209 (T209R) in the ectodomain of DR4 (Fig. 2)
. Eleven (35%) of the lung cancer cell lines exhibited only the altered allele. Analysis of matched normal DNA from the same patients revealed that 7 were homozygous and 4 were heterozygous for the alteration. Of the 10 tumors heterozygous for the C626G allele, 9 of their matched normals were also heterozygous, whereas 1 displayed only the common allele (change subsequently acquired in the tumors). Of the 10 tumors without the C626G change, 5 of the matched normals were found to be heterozygous for the alteration (Table 2)
. These 5 samples have all been previously noted to have deletions at 8p21 (10)
. Of note, there was no difference in the frequency of the C626G allele between the SCLC and NSCLC cell lines.
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. The frequency of being homozygous for C626G (35%) was significantly increased in patients with lung cancer as compared with the normal volunteers (2 = 4.1, P = 0.044). Because it is possible that racial differences between cases and normal volunteers could influence the results, we repeated the analysis with a race-matched control group. Evaluation of these 45 normal control patients matched for age and race (group 3; Table 2
) revealed that 10 (22%) controls were homozygous for the altered allele. Although the frequency of homozygosity for C626G was also elevated in lung cancer cell lines relative to matched controls, this was no longer statistically significant.
Subsequent evaluation of 21 primary NSCLC tumors revealed 10 (48%) specimens homozygous for the C626G alteration, which was significantly increased relative to the age- and race-matched controls (2 = 4.4, P = 0.037; Table 3
). The matched normal DNA for all of the 10 specimens was also homozygous for the C626G change. Of the other 11 tumor samples, 5 were heterozygous and 6 had only the wild-type allele (Table 2)
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and 3
). The frequency of individuals homozygous for C626G was elevated relative to age- and race-matched controls (2 = 4.0, P = 0.044; Table 3
). Of the gastric adenocarcinoma specimens, 11 of 25 (44%) were homozygous for the C626G alteration. Although the frequency of C626G homozygosity in the gastric adenocarcinoma samples was increased relative to the matched (group 3) controls, this did not reach statistical significance (2 = 3.6, P = 0.057).
Identification of a G422A Allele in Exon 3 of TRAIL Receptor DR4.
Sequence analysis of exon 3 in 31 lung cancer cell lines revealed that 11 (35%) were homozygous for a missense alteration at nucleotide 422 changing a guanine to adenine (G422A), resulting in a substitution of a histidine for arginine at codon 141 (R141H) in the ectodomain of DR4 (Tables 4
and 5
). Analysis of matched, normal DNA was similar to that of the C626G allele, because the C626G and G422A allele status was identical in all of the 31 lung cancer cell lines.
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Subsequent evaluation of the 21 primary NSCLC, 19 HNSCC, and 25 gastric adenocarcinoma samples revealed that 48, 47, and 48%, respectively, were homozygous for the G422A alteration, whereas only 13% of the 45 age- and race-matched control samples were homozygous (Table 4)
. In comparison with the matched controls, the frequency of G422A homozygosity was significantly elevated in all of the three primary tumor groups (2 = 9.2, P = 0.002 for NSCLC; 2 = 8.6, P = 0.003 for HNSCC; and 2 = 10.1, P = 0.001 for gastric adenocarcinoma) and in the lung cancer cell lines (2 = 5.2, P = 0.023; Table 5
).
Evaluation of both the C626G and G422A alleles revealed that they segregated together in 96% of all of the samples (n = 243) evaluated (tumor and normal). In only 9 cases (2 gastric adenocarcinoma samples and 7 matched controls) was the C626G and G422A allele status different. All of the lung cancer cell lines and the primary NSCLC and HNSCC samples that expressed the G422A homozygosity also expressed the C626G homozygosity. All but 1 of the gastric adenocarcinoma specimens expressing G422A homozygosity also expressed the C626G homozygosity. The frequency of being homozygous for both alleles was significantly elevated in lung cancer cell lines as well as primary NSCLC, HNSCC, and gastric adenocarcinoma samples relative to matched controls (group 3; Table 6
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have identified a missense alteration at nucleotide 626 of DR4 that changes a cytosine to a guanine and results in a substitution of an arginine for threonine at codon 209 (T209R). This altered allele, although present in a homozygous state in only 22% of age- and race-matched controls, was noted at a frequency of 35, 48, 47, and 44% in lung cancer cell lines and primary NSCLC, primary HNSCC, and gastric adenocarcinoma samples, respectively. We have also identified a missense alteration at nucleotide 422 of DR4 that changes a guanine to an adenine and results in a substitution of histidine for arginine at codon 141 (R141H). This altered allele was found at a frequency of 35, 48, 47, and 48% in lung cancer cell lines and primary NSCLC, primary HNSCC, and gastric adenocarcinoma samples, respectively, but was present homozygously in only 13% of matched controls. DR5 mutations described previously occurred at a frequency of <11% in tumor samples (17 , 18) .
It is quite possible that some of the "homozygotes" identified in our tumor samples may actually be hemizygous (have only one allele present). Our assays did not allow us to unambiguously distinguish these two possibilities. 8p21 deletions are frequent events in both lung cancer and HNSCC. Although the 8p21 status of our primary tumors is unknown, the majority of the lung cancer cell lines studied here have large areas of 8p21 deletion (10) . In fact, 8 of the 11 lung cancer cell lines "homozygous" for the C626G and G422A alleles have known 8p21 deletions, whereas 3 do not exhibit allelic losses in this area (10) . Functionally, the presence of either one or two copies of an altered allele in the absence of any wild-type alleles would be expected to be equivalent. In addition, there were 7 tumor samples (4 lung cancer cell lines and 3 gastric adenocarcinomas) homozygous and 7 tumor samples (5 lung cancer cell lines and 2 gastric adenocarcinomas) wild type for both alterations, the matched normal DNA of which was heterozygous. This result is likely explained by LOH, because all 9 of these lung cancer cell lines have known 8p21 deletions, and it is quite possible that the gastric adenocarcinoma specimens do as well. One possible interpretation of these results is that the C626G and G422A alleles may predispose to cancer under certain conditions, but once cancer develops they may not be required for tumor maintenance.
The significantly increased frequency of homozygosity for both alleles and the G422A allele individually in all of the four tumor groups relative to matched controls, as well as the significantly increased frequency of C626G homozygosity in primary NSCLC and HNSCC, suggests that these alleles are more than silent polymorphisms. In addition, the C626G and G422A alleles were not present homozygously in the germ-line of any matched normal sample, the tumor of which displayed only the wild-type allele. In addition, although both alterations appear to be germ line in nature, analysis of matched normal DNA reveals that the alteration occurred somatically in at least 1 case.
Although it is accepted that tumorigenesis is the result of accumulation of many genetic alterations, the frequency of the C626G and G422A alleles in tumor samples and their occurrence as both a somatic and germ-line alteration suggest that, at a minimum, the altered alleles may be a marker for predisposition to lung cancer, HNSCC, and gastric adenocarcinoma. Genetic alterations may change the susceptibility of a cell for cancer. Environmental influences may interact with genetic predispositions to contribute to tumorigenesis. Smoking is a well-defined risk factor for lung cancer (1) . Perhaps one or both of these alterations confer an increased risk of development of lung cancer on heavy smokers as compared with light smokers or nonsmokers. This is a hypothesis that may be further investigated in a larger case control study.
Recently, the crystal structure of the TRAIL/DR5 complex has been described (26)
. Two main interface regions were identified, the 50’s loop and 90’s loop. DR4 has 69% sequence identity with DR5 in its cysteine-rich, ligand-binding motif (Fig. 4)
. The C626G alteration changes a threonine to an arginine (charge change) immediately 3' to one of these two main interface regions between TRAIL and its proapoptotic receptors. The G422A alteration causes an amino acid change (arginine to histidine) just 5' to the ligand-binding domain of DR4. This suggests that one or both of these alterations may alter TRAIL binding to DR4 and, perhaps, result in deficient apoptotic signaling.
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FOOTNOTES |
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1 Supported in part by a Hematology-Oncology Training Grant to the Children’s Hospital of Philadelphia (to M. J. F.). W. S. E-D. is an Assistant Investigator of the Howard Hughes Medical Institute. 
2 To whom requests for reprints should be addressed, at Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, 415 Curie Boulevard, CRB 437A, Philadelphia, PA 19104. Phone: (215) 898-9072; Fax: (215) 573-9139; E-mail: wafik{at}mail.med.upenn.edu
3 The abbreviations used are: SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; HNSCC, head and neck squamous cell cancer; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; DD, death domain; LOH, loss of heterozygosity.
Received 12/ 8/00; revised 2/28/01; accepted 3/ 2/01.
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B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity, 7: 813-820, 1997.[CrossRef][Medline]
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