Clinical Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Y.-C.
Right arrow Articles by Lin, C.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Y.-C.
Right arrow Articles by Lin, C.-C.
Clinical Cancer Research Vol. 12, 2716-2729, May 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Genomic Alterations in Human Malignant Glioma Cells Associate with the Cell Resistance to the Combination Treatment with Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand and Chemotherapy

Yueh-Chun Li1, Ching-Cherng Tzeng3, Jin H. Song4, Fuu-Jen Tsia2, Lie-Jiau Hsieh2, Shu-Ju Liao2, Chang-Hai Tsai2, Erwin G. Van Meir4, Chunhai Hao4,5 and Chyi-Chyang Lin2,5

Authors' Affiliations: 1 Department of Biomedical Sciences, Chung Shan Medical University; 2 Department of Medical Research, China Medical University and Hospital, Taichung, Taiwan; 3 Department of Pathology and Laboratory Medicine, Chi-Mei Foundation Medical Center, Tainan, Taiwan; 4 Departments of Pathology and Laboratory Medicine, Neurosurgery and Whinship Cancer Institute, Emory University, Atlanta, Georgia; and 5 Department of Laboratory Medicine and Pathology, University of Alberta, Alberta, Canada

Requests for reprints: Chyi-Chyang Lin, Department of Medical Research, China Medical University and Hospital, 2. Yuh-Der Road, 404 Taichung, Taiwan. Phone: +886-4-22053366; Fax: +886-4-22033295; E-mail: lincc{at}mail.cmu.edu.tw.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is currently under clinical development as a cancer therapeutic agent. Many human malignant glioma cells, however, are resistant to TRAIL treatment. We, therefore, investigated the genomic alterations in TRAIL-resistant malignant glioma cells.

Experimental Design: Seven glioma cell lines and two primary cultures were first analyzed for their sensitivity to TRAIL and chemotherapy and then examined for the genomic alterations in key TRAIL apoptotic genes by comparative genomic hybridization (CGH), G-banding/spectral karyotyping, and fluorescence in situ hybridization (FISH).

Results: CGH detected loss of the chromosomal regions that contain the following genes: 8p12-p23 (DR4 and DR5), 2q33-34 (caspase-8), 11q13.3 (FADD), 22q11.2 (Bid), and 12q24.1-q24.3 (Smac/DIABLO) in TRAIL-resistant cell lines. Spectral karyotyping showed numerical and structural aberrations involving the chromosomal regions harboring these genes. A combination of G-banding/spectral karyotyping and FISH further defined the loss or gain of gene copy of these genes and further showed the simultaneous loss of one copy of DR4/DR5, caspase-8, Bid, and Smac in two near-triploid cell lines that were resistant to the combination treatment with TRAIL and chemotherapy. Loss of the caspase-8 locus was also detected in a primary culture in correlation with the culture resistance to the combined TRAIL and chemotherapy treatment.

Conclusions: The study identifies chromosomal alterations in TRAIL apoptotic genes in the glioma cells that are resistant to the treatment with TRAIL and chemotherapy. These genetic alterations could be used to predict the responsiveness of malignant gliomas to TRAIL-based therapies in clinical treatment of the tumors.

Malignant gliomas are the most common and most aggressive human brain tumors, and there is no curative treatment (1). Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) of the tumor necrosis factor family (2, 3) has been shown to induce apoptosis in malignant glioma cells in cultures and experimental animals (4–6) and thus is currently under development as a cancer therapeutic agent. However, malignant gliomas show heterogeneity in their response to TRAIL; some malignant glioma cells are TRAIL sensitive, whereas others are resistant (5, 7, 8). To overcome TRAIL resistance, chemotherapeutic agents have been investigated for their synergistic effects with TRAIL in malignant glioma cells (4, 7, 9, 10). In this study, however, we have identified malignant glioma cell lines that are resistant to the combined TRAIL and chemotherapy treatment. To explore the genetic mechanisms in the cell resistance, we have studied malignant glioma cells for their genomic alterations involving the gene loci encoding the TRAIL apoptotic signaling pathways.

TRAIL induces apoptosis through both receptor-mediated extrinsic and mitochondria-involved intrinsic pathways (11). TRAIL interacts with its death receptors DR4 and DR5 to recruit intracellular Fas-associated death domain (FADD; ref. 12) and caspase-8 (13) for the assembly of a death-inducing signaling complex (14). In the death-inducing signaling complex, caspase-8 is cleaved and then initiates apoptosis through cleavage of downstream caspase-3 (15). However, malignant glioma cells express X-linked inhibitor of apoptosis protein (16) that interacts with caspase-3 and inhibits caspase-8 cleavage of caspase-3 (17). To rescue this extrinsic pathway, caspase-8 cleaves Bcl-2 inhibitory BH3 domain–containing protein (Bid; ref. 18) that in turn induces mitochondrial release of second mitochondria-derived activator of caspase/direct inhibitor of apoptosis binding protein with low isoelectric point (Smac/DIABLO; refs. 19, 20). In the cytosol, Smac interacts with X-linked inhibitor of apoptosis protein to release its inhibition of caspase-3 (17). Caspase-3 subsequently cleaves its substrates, such as caspase-7 (21) and DNA fragmentation factor 45 (22), in the execution of the programmed cell death.

Recent advances in molecular cytogenetic techniques allow the identification of complex chromosomal abnormalities in malignant gliomas (23–25). Here, we apply comparative genomic hybridization (CGH; ref. 26), spectral karyotyping (27), and fluorescence in situ hybridization (FISH) with chromosome region specific probes in a systemic analysis of malignant glioma cell lines for their genomic alterations in the chromosome regions that harbor key TRAIL apoptotic genes: 8p12-p23 (DR4 and DR5; refs. 28, 29), 11q13.3 (FADD; ref. 30), 2q33-q34 (caspase-8; ref. 31), 4q34-q35 (caspase-3; ref. 32), 10q25.1-q25.2 (caspase-7; ref. 32), 1p36.1-p36.3 (caspase-9; ref. 33), 22q11.2 (Bid; ref. 34), and 12q24.1-q24.3 (Smac/DIABLO; ref. 19). The results presented here show the genomic alterations in the chromosomal regions that contain DR4/DR5, caspase-8, FADD, Bid, and Smac loci in the cell lines resistant to the combination treatment with TRAIL and chemotherapy.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and cell death assay. The human malignant glioma cell lines LN71, LN215, LN443, LN464 (35), U118MG, U138MG, and U373MG (American Type Culture Collection, Rockville, MD) were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen). Early passage of primary malignant glioma cultures were established from fresh operative glioblastoma tumors and cultured in DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mmol/L L-glutamine, and 1 mmol/L sodium pyruvate as previously reported (36). For cell death analysis, cell lines were seeded in 96-well plates at 3 x 105 per well and treated with the recombinant human TRAIL (114-281 amino acids, 19.6 kDa; PeproTech, Inc., Rocky Hill, NJ), along or in combination with each of the following chemotherapeutic agents: camptothecin, cisplatin, doxorubicin, etoposide (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada), and temozolomide (kindly provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD). The chemotherapeutic agents were dissolved in DMSO (Sigma-Aldrich Canada). Cell death was determined by the crystal violet assay (5), and the results were presented as the percentage cell death: 1 – (absorbance of cells treated / absorbance at 550 nm of cells untreated) x 100 (7).

Caspase cleavage and Western blots. The cells were lysed in lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 1% Triton X-100, 1% protease inhibitor mixture, and 1 mmol/L phenylmethylsulfonyl fluoride). Fifty micrograms of total proteins from each lysate were separated through 15% SDS-PAGE and transferred to nitrocellulose membranes. For caspase cleavage, the membranes were incubated overnight at 4°C with mouse monoclonal anti-human caspase-8 (Medical and Biological Laboratories, Nagoya, Japan) and rabbit anti-human/caspase-3, DNA fragmentation factor 45 (StressGen, Victoria, British Columbia, Canada), caspase-7, and caspase-9 (ProSci., Inc., Poway, CA). For protein expression, the membranes were incubated with mouse monoclonal anti-human FADD, cytochrome c, and X-linked inhibitor of apoptosis protein (Transduction Laboratories, Lexington, KY); rabbit anti-human DR4 and DR5 (ProSci.); Bid (Biosource International, Inc., Camarillo, CA), and extracellular signal-regulated kinase 1/2 (StressGen). The membranes were incubated for 1 hour with horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit antibody and developed by chemiluminescence (Amersham Biosciences, Piscataway, NJ).

CGH. CGH was carried out based on the protocol as previously described (37). In brief, the test DNA and reference DNA were labeled with fluorescein-12-dUTP and Texas red-5-dUTP (NEN Life Science, Boston, MA), by nick translation respectively. Four hundred nanograms of labeled test DNA, 400 ng of labeled reference DNA, and 10 µg of unlabeled Cot1 DNA (Invitrogen) were then mixed in 10 µL of hybridization solution (70% formamide, 10% dextran sulfate), denatured at 70°C for 5 minutes, and hybridized to target metaphase slides at 37°C for 3 days. Before hybridization, the slides were denatured in 70% formamide, 2 x SSC [0.3 mol/L NaCl, 30 mmol/L Na3 citrate (pH 7)] at 74°C for 3 minutes and dehydrated. After hybridization, slides were washed twice with 50% formamide, 2 x SSC solution at 45°C, once with 2 x SSC, and once with PN buffer (0.1 mol/L NaB2BHPOB4B/NaHB2BPOB4B with 0.1% NP40) at room temperature. The slides were counterstained with 4',6-diamidino-2-phenylindole (200 ng/mL in 2 x SSC) in an anti-fade solution (Vector Laboratories, Burlingame, CA).

Only slide batches showing high-intensity and uniform hybridization were used for imaging analysis. Red, green, and blue images from representative metaphase spreads were digitalized under a fluorescence microscope equipped with a CytoVision imaging system (Applied Imaging, Santa Clara, CA). Karyotypes from 12 to 15 metaphases were combined to generate a mean CGH ratio profile for every hybridization experiment. Detection of chromosomal imbalances was done using Applied Imaging CytoVision High-Resolution CGH software (38).

G-banding and spectral karyotyping. G-banding karyotyping was done according to the standard procedure. Spectral karyotyping analysis was done on destained G-banded slides or unstained slides that were aged for 2 days after fresh preparation. Human SkyPaint kits containing 24 color chromosome-specific painting probes, hybridization buffer, and 4',6-diamidino-2-phenylindole counterstain (Applied Spectral Imaging Ltd., Migdal Haemek, Israel) were used following the manufacturer's instructions. Briefly, slides were denatured in 70% formamide, 2 x SSC at 75°C for 5 minutes. SkyPaint probes were denatured at 80°C for 10 minutes, preannealed at 37°C for 1 hour, and hybridized to denatured slides for 36 to 48 hours in a 37°C humidified chamber. After hybridization, the 24 colored chromosome images were captured with an SD 300-VDS Cytogenetic Workstation (Applied Spectral Imaging) equipped with a Leica DM LB FLUO microscope and analyzed using the ASI SkyView version 1.6 software. Simultaneous pseudocolor chromosome images were created to differentiate chromosome regions of similar color involved in structural rearrangements. Composite karyotypes based on 12 to 25 G-banding/spectral karyotyping karyotypes were constructed.

FISH. The DNA probes included DR5 cDNA in P1 clone (39) and the chromosome region–specific BAC clones (CHORI BAC/PAC Resources) for caspase-8 (RPCI11-575C6), caspase-3 (RPCI11-367I6), caspase-7 (RPCI11-211N11), Bid (RPCI11-91O6), and Smac/DIABLO (RPCI11-512M8). Centromere DNA-specific probes, including gamma 8 (40), CEP 2, CEP 5, CEP 10, CEP 12, and CEP18 (Vysis, Inc., Des Plaines, IL), and chromosome region–specific clones RP11-268N2 (CHORI BAC/PAC Resources), DiGeorge N25 (D22S75; Vysis), or P109L3 (41) were used for chromosome identification. DNA was isolated and labeled with SpectrumGreen-dUTP or SpectrumRed-dUTP (Vysis) by nick translation. The procedures of denaturation, hybridization, and signal detection labeled probe for FISH analysis were previously described (40, 42). Hybridization signals were captured and analyzed using a FISH workstation (Perceptive Scientific Instruments, Inc., League City, TX) with MacProbe v4.0 software.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Resistance of malignant glioma cell lines to TRAIL and chemotherapy. We have examined a large number of human malignant glioma cell lines for their sensitivity to TRAIL (5, 7). Six TRAIL-resistant malignant glioma cell lines (LN443, U138MG, U118MG, LN464, LN215, and U373MG) and one TRAIL-sensitive line (LN71) were further examined in this study. First, the cell lines were treated with 300 ng/mL of TRAIL for 16 hours, and cell death analysis further confirmed LN443, U138MG, U118MG, LN464, LN215, and U373MG cell lines were TRAIL resistant (Fig. 1A ). Next, we examined if chemotherapeutic agents camptothecin, cisplatin, doxorubicin, etoposide, and temozolomide can enhance TRAIL killing of these TRAIL-resistant glioma cell lines. In the presence of cisplatin, four of the six TRAIL-resistant cell lines (U118MG, U138MG, LN443, and LN464) became TRAIL sensitive (Fig. 1A), whereas two cell lines (LN215 and U373MG) remained resistant to the combination treatment (Fig. 1A). These two cell lines were further treated with TRAIL in combination with camptothecin, doxorubicin, etoposide, or temozolomide. The results showed that none of these chemotherapeutic agents enhanced TRAIL killing of these two resistant cell lines (Fig. 1B). This study, therefore, identified human malignant cells that are resistant to the combination treatment of TRAIL and chemotherapy. Based on this study, we generally reclassified the resistant cell lines into TRAIL resistant (U118MG, U138MG, LN443, and LN464) and TRAIL/chemotherapy resistant (LN215 and U373MG).


Figure 1
View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1. TRAIL sensitivity and expression of key TRAIL apoptotic genes in human malignant glioma cell lines. A, each of the seven cell lines was grown in 96-well plates (3 x 104 per well) and treated with 300 ng/mL TRAIL, alone or in combination with 10 µg/mL cisplatin (Cis) for 16 hours. Cell death was determined by a crystal violet assay. Columns, mean U (n = 6); bars, SE. B, LN215 and U373MG cell lines were treated with the combination of 300 ng/mL TRAIL and 10 µg/mL camptothecin (CPT), cisplatin, doxorubicin (Dox), etoposide (Eto), and temozolomide (TMZ) for 16 hours and analyzed by cell death analysis. C, TRAIL-induced cleavage of caspases. Each of the cell lines was treated with 300 ng/mL TRAIL in the absence and presence of 10 µg/mL cisplatin for the time indicated. Left, cell lysates were subjected to Western blot analysis using antibody to caspase-8 (Casp-8), caspase-9 (Casp-9), caspase-7 (Caps-7), caspase-3 (Casp-3), and DNA fragmentation factor 45 (DFF45). D, expression of TRAIL apoptotic proteins in seven glioma cell lines as detected by Western blots with extracellular signal-regulated kinase 1/2 (ERK1/2) as protein loading control.

 
To investigate mechanisms in the cell resistance to TRAIL and chemotherapy, we first examined TRAIL-induced cleavage of caspases in these glioma cell lines. A TRAIL-sensitive line (LN71), a TRAIL-resistant line (LN443), and two TRAIL/chemotherapy-resistant lines (LN215 and U373MG) were treated with TRAIL, alone or in combination with cisplatin, and subjected to Western blots. The cleavage products of caspase-8, caspase-9, caspase-3, caspase-7, and DNA fragmentation factor 45 were detected in TRAIL-sensitive LN71 treated with TRAIL (Fig. 1C) and TRAIL-resistant LN443 cell line after the combination treatment with TRAIL and cisplatin (Fig. 1C). In contrast, no cleavage products of these caspases were detected in TRAIL/chemotherapy-resistant LN215 and U373MG cell lines regardless of the presence of cisplatin (Fig. 1C). We then examined the expression of key TRAIL apoptotic proteins in these cell lines. DR5 and caspase-8 were expressed at consistent levels in the TRAIL-sensitive (LN71) and TRAIL-resistant cell lines (LN443, U138MG, U118MG, and LN464); however, the expression of these proteins was significantly inhibited in TRAIL/chemotherapy-resistant LN215 and U373GM cell lines (Fig. 1D). FADD was consistently expressed in all the cell lines, whereas Bid expression was down-regulated in TRAIL-resistant and TRAIL/chemotherapy-resistant lines except LN464 (Fig. 1D). Smac expression seemed slightly lower in TRAIL/chemotherapy-resistant cell lines. Caspase-3 was consistently expressed in all the cell lines, and caspase-7 and caspase-9 expression seemed to be slightly higher in the resistant than the sensitive line (Fig. 1C). The study suggests that inhibition of the expression of key TRAIL apoptotic genes may result in glioma cell resistance to TRAIL and chemotherapy treatment. We, therefore, investigated whether genomic alterations were present in these key TRAIL apoptotic genes that could explain the inhibition of the gene expression.

CGH detection of genomic imbalances with key TRAIL apoptotic gene regions. CGH was done on seven malignant glioma cell lines, and the genomic imbalances detected were summarized in Fig. 2 . Attempts were made to identify loss or gain of the chromosomal regions that harbor key TRAIL apoptotic genes: 8p12-p23 (DR4 and DR5; refs. 28, 29), 11q13.3 (FADD; ref. 30), 2q33-q34 (caspase-8; ref. 31), 4q34-q35 (caspase-3; ref. 32), 10q25.1-q25.2 (caspase-7; ref. 32), 22q11.2 (Bid; ref. 34), 19q13.3-q13.4 (Bax; ref. 43), 6p21.2-p21.3 (Bak; ref. 44), and 12q24.1-q24.3 (Smac; ref. 19). The CGH analysis revealed genomic imbalances in the chromosomal regions that harbor DR4/DR5, FADD, caspase-3, caspase-7, caspase-8, caspase-9, Bid, and Smac loci in the cell lines; however, multiple losses involving these regions were more commonly found in TRAIL-resistant cell lines. Specifically, loss of the region 2q33-q34 (caspase-8) was observed in the TRAIL/chemotherapy-resistant LN215, whereas TRAIL-resistant LN464 and U138MG and TRAIL-sensitive line LN71 showed a gain in this region. Losses of the region 8p12-p23 (DR4/DR5) and the region 22q11.2 (Bid) were observed in the two TRAIL/chemotherapy-resistant lines LN215 and U373MG. Loss of the region 4q34-q35.1 (caspase-3) was detected in the TRAIL/chemotherapy-resistant U373MG and the TRAIL-resistant LN464. The TRAIL-resistant line U118MG and the TRAIL-sensitive line LN71 showed loss involving a large portion of chromosome 4 that may extend to the 4q33-q35 region. Except for U373MG, all the TRAIL-resistant and TRAIL/chemotherapy-resistant cell lines showed the loss of the chromosomal region 10q25.1-q25.2 (caspase-7). Loss of the chromosomal region 11q13.3 (FADD) was found in two TRAIL-resistant lines LN443 and LN464. The two TRAIL/chemotherapy-resistant lines and two TRAIL-resistant lines (LN464 and LN443) exhibited loss of part of chromosome 12, including the region 12q24.1-q24.3 (Smac). None of these losses were detected in the TRAIL-sensitive cell line LN71. On the other hand, loss of the chromosome region 1p36.1-p36.3 (caspase-9) was only detected in this sensitive line.


Figure 2
View larger version (39K):
[in this window]
[in a new window]
  		Fig. 2. A summary of CGH profiles showing genomic imbalance in seven human malignant glioma cell lines. Gains of chromosomal regions are represented by vertical bars on the right hand side of the chromosome ideogram, whereas loss of chromosome regions are represented by the vertical bars on the left of the chromosome ideogram. TRAIL-sensitive (pink bars), TRAIL-resistant (orange bars), and TRAIL/chemotherapy-resistant cell lines (blue bars). Loss or gain of eight particular chromosome regions associated with key TRAIL apoptotic genes were observed (green horizontal bars and arrows).

 
G-banding/spectral karyotyping identification of chromosome aberrations. CGH can identify losses and gains of chromosomal regions (26), whereas the combination of G-banding and spectral karyotyping allows the detection of cryptic and complex structural abnormalities (45). The cell lines were, therefore, subjected to G-banding/spectral karyotyping, and the composite G-banding/spectral karyotyping karyotypes were constructed based on 11 to 26 metaphase spreads from each of the cell lines (Table 1 ). All the cell lines examined were near polyploidy from 3n– to 6n– with chromosome numbers ranging from 62 to 68 in LN215 to 133 to 135 in LN443. Multiple chromosome aberrations were found in each cell line, with structural aberrations varying from 8 in U373MG to 36 in LN464. The composite karyotypes also highlighted the heterogeneity of each of the cell lines and identified the number of cells examined with loss, gain, or retention of specific intact chromosomes or structural aberrant chromosomes that harbor key TRAIL apoptotic gene loci (Table 2 ).


View this table:
[in this window]
[in a new window]
  		Table 1. Composite karyotypes of seven glioma cell lines based on the results of G-banding/spectral karyotyping analyses

 

View this table:
[in this window]
[in a new window]
  		Table 2. Summary of chromosome aberrations involving regions harboring key TRAIL apoptotic gene loci in seven glioma cell lines identified by G-banding/spectral karyotyping

 
G-banding/spectral karyotyping revealed multiple structural aberrations in the regions that harbor TRAIL apoptotic genes (Fig. 3 ). The TRAIL-sensitive cell line LN71 has a near tetraploid karyotype with a chromosome number varying from 80 to 87 in 18 metaphase spreads examined. In contrast, the TRAIL/chemotherapy-resistant LN215 and U373MG are near triploid. These two TRAIL/chemotherapy-resistant lines showed simultaneous losses of chromosomes or chromosomal regions that involved DR4/DR5, caspase-8, Bid, and Smac loci in the majority of cells examined. For example, 77% (10 of 13), 100% (13 of 13), 92% (12 of 13), and 77% (10 of 13) of LN215 cells studied showed losses of chromosomes 8, 2, 22, and 12, respectively (Table 1). An isochromosome [i(8)(q10)] with the loss of 8q21-q22 (DR4/DR5) was found in 95% (19 of 20) of U373MG cells examined. U373MG cells also showed losses of chromosomes 12 and 22 in 80% (16 of 20) and 95% (19 of 20) of the cells analyzed, and about 30% (7 of 20) cells studied were missing of a chromosome 2 that contains the caspase-8 locus. No loss of chromosome or chromosomal region involving caspase-8, Bid, and Smac was detected in the near-tetraploid TRAIL-sensitive line LN71.


Figure 3
View larger version (44K):
[in this window]
[in a new window]
  		Fig. 3. Partial G-banding/spectral karyotyping karyotypes with structural aberrant chromosomes selected from Table 2 in seven glioma cell lines. Structural aberrant chromosome that associates with loss, retaining, or gain of a specific key TRAIL gene is indicated by bold italic, italic, and bold character referred in Table 2.

 
Losses of chromosomes or chromosomal regions associated with the abovementioned four chromosomes were also found in near-triploid TRAIL-resistant lines. However, some of the losses were compensated by structural aberrations that have a gain or retention of the gene loci. In LN443, a derivative chromosome, der(8)t(8;20)(p11.2;p11.2) had lost the DR4/DR5 region 8p21-p22. In U138MG, loss of a chromosome 8, which contains the DR4/DR5 region, was observed. Loss of a chromosome 2 that contains the caspase-8 region (2q33-q34) was found in >50% (7 of 13) of LN443 cells, but retention of this caspase-8 locus was also observed in a derivative chromosome, der(2)t(2;7)(q37;q22), from this cell line. Both LN443 and U138MG had lost the chromosome 22 involving the Bid locus, but this was compensated by an idic(22)(p11.2) and a der(22)t(5;22)(q15;p11.2) that has a gain or retention of the Bid region, respectively. Each of three structural aberrant chromosomes [dic(12;18)(q10;q10), der(12)t(1;12)(p31.2;q15), and der(12)t(5;12)(q33;q24.3)del(5)(q35)] with loss of the Smac/DIABO region 12q24.3 was also found in the majority of LN443, U138MG, and U118MG cells. However, 45% (5 of 11) of LN443 cells had a derivative chromosome der(3;12)(q10;q10), and 92% (11 of 13) of LN464 cells had a dicentric chromosome [dic(12;16)(9p13;q12)]. Both aberrant chromosomes had retained the Smac locus.

As for the FADD locus, neither loss of chromosome 11 nor structural aberration involving the chromosome region 11q13 was observed in the TRAIL-sensitive cell line LN71. However, a breakage at the region 11q13 was found in three different derivative chromosomes [der(11)t(11;15)(q13;q15), der(15)t(2;15)(p21;p11.2)t(11;15)(q13;q15), and der(15)t(11;15)(q13;q15)] in TRAIL-resistant LN464. Among these derivative chromosomes, the first one lost the region with the FADD locus. The remaining two had a gain of the region associated with the locus. A derivative chromosome [der(11)t(11;19)(p15;p13.2)] and a deleted chromosome 11 [del(11)(q14)] found in >89% of U138MG and 45% of LN443 cells, respectively, both retained the region with the FADD locus. Over 50% of the cells examined in the TRAIL/chemotherapy-resistant cell line U373MG had a derivative chromosome [der(11)del(11)(q12)t(10;11)(q26;p11.2)], which showed loss of the FADD region.

G-banding/spectral karyotyping analysis also detected specific chromosome aberrations involving loss, gain, or retention of caspase-3, caspase-7, and caspase-9 regions 4q35, 10q25.1, and 1p36.1-p36.3, respectively. However, the chromosome aberrations were found to occur in both TRAIL-resistant and TRAIL-sensitive cell lines. For example, loss of a chromosome 4 that harbored the caspase-3 locus was found in TRAIL/chemotherapy-resistant lines (LN215 and U373MG), TRAIL-resistant lines (LN443 and LN464), and TRAIL-sensitive line (LN71). Loss of a chromosome 10 containing the caspase-7 locus was observed in the majority of cells studied from all the cell lines. Structural aberrant chromosomes involving loss of the caspase-9 region 1p36.1-p36.3 were found in all cell lines studied except the TRAIL/chemotherapy-resistant LN215, which had a derivative chromosome der(1;5)(q10;q10) with loss of the caspase-9 region in <50% of the cells examined and also had a derivative chromosome der(1;5)(p10;q10) that retained the caspase-9 region.

Combined G-banding/spectral karyotyping and FISH analyses of gene dosages. The composite karyotypes presented in Table 1 did not include the copy number for each type of numerical or structural aberrant chromosomes because we observed variable copy numbers of those aberrant chromosomes occurring in different cells examined from the same cell line. In addition, some cryptic rearrangements involving a gene locus of interest could also escape the detection by G-banding and spectral karyotyping analyses. This could affect the end result of estimation for the total gain or loss of chromosomal region for a particular locus from this line. However, a structural aberration that has lost, gained, or retained a specific key TRAIL gene region can be verified by FISH with chromosome region–specific probes. Therefore, we further examined the gene copy numbers, which involve the key TRAIL genes by a combination of G-banding/spectral karyotyping and FISH analysis (Table 3 ).


View this table:
[in this window]
[in a new window]
  		Table 3. Summary of modal gene copy number of eight key TRAIL-induced apoptotic gene loci in seven glioma cell lines determined by G-banding/spectral karyotyping and FISH analyses

 
This combined G-banding/spectral karyotyping and FISH analysis showed that 77% (10 of 13) of the 3n– LN215 cells (TRAIL/chemotherapy resistant) only had two copies of the DR5 locus either due to loss of a chromosome 8 or having it replaced by an isodicentric chromosome 8 lacking the DR5 region as identifying with the DR5 region–specific probe by FISH (Fig. 4A ). This resulted in the net loss of one copy of the DR5 locus in LN215 cells. The other TRAIL/chemotherapy-resistant line U373MG also lost one copy of the DR5 region in 95% cells examined due to the replacement of one normal chromosome 8 by an isochromosome i(8)(q10). This isochromosome was negative for FISH signal with the DR5 region probe; thus, those cells examined only exhibited two DR5 signals (Fig. 4B), indicating loss of one DR5 copy. The similar loss of a DR5 copy was observed in 68% 3n– TRAIL-resistant cell line U138MG. The other TRAIL-resistant lines either had no loss of the region, such as in LN464 that had a chromosome 8 with an inversion involving the 8p21-p22 region, and FISH study confirmed no loss of the DR5 region (Fig. 4C) in the majority of the cells (77%) examined, or only a small portion (<50%) cells examined having loss of the locus as in U118MG and LN443. Loss of one copy of the chromosome 8 with DR5 locus was also found in 83% of cells examined from 4n TRAIL-sensitive line LN71, and the significance of this finding will be discussed later.


Figure 4
View larger version (43K):
[in this window]
[in a new window]
  		Fig. 4. Chromosome mapping of DR5, caspase-8, Bid, Smac, and caspase-3 genes by FISH analysis. A to C, dual hybridization with a P1 clone DNA probe for the DR5 gene labeled with red fluorescence and gamma 8 centromeric satellite DNA probe (for identification of chromosome 8) labeled with green fluorescence was done on metaphase spreads. A, metaphase of LN215 (3n–) shows only two chromosome 8s bearing both DR5 signal (red) and the centromere 8 signal (green). An isodicentric 8q chromosome (arrow) shows loss of DR5 signal. B, metaphase of U373MG (3n–) shows two chromosome 8s both with red and green signals and an isochromosome 8q, which only has a green signal of the chromosome 8 centromere (arrow). C, A LN464 (4n) cell shows three chromosome 8s each with both DR5 (red) and centromere 8 signals (green) and an inverted chromosome 8 [inv(8)(p11.2p21)], which the DR5 sequence is located close to the centromeric region and shows the yellow signal (yellow arrow). D to F, dual hybridization with BAC clone probe for caspase-8 (RPCI11-575C6; green fluorescent) and centromeric probe for chromosome 2 (CEP 2, Vysis; red fluorescent) was done on the metaphase spreads. A LN215 cell (D) and a LN443 cell (E) both show only two chromosome 2s having green and red signals. F, A LN464 metaphase cell shows four chromosome 2s with both green and red hybridization signals, a dicentric chromosome dic(2;19)(q31;q12) (arrow), which retains the centromere of the chromosome 2 (red signal) but lacks green signal. G and H, dual hybridization was carried out with a BAC clone probe for the Bid gene (RPCI11-91O6; red) and the Vysis probe for LSI DiGeorge N25 region (22q11.2; green) as control. A 3n– LN215 cell (G) and a 3n– U373MG cell (H) show only two chromosome 22s having cohybridization of red and green signals in each chromosome 22s of the cells. A 4n– LN464 cell (I) shows a chromosome 22 having both green and red signals closely associated (indicated), and three structural aberration chromosomes (listed in Table 3) each retains the Bid region and DiGeorge region (arrows). J to L, dual hybridization with BAC clone probe RPCI11-512M8 for Smac locus (red) and control probe CEP12 (green) was conducted on glioma cell lines. Only two chromosome 12s that have the Smac locus (red) and the control signal (green) are seen in a 3n– cell of LN215 (J) and a 3n– cell of U373MG (K). L, metaphase of the 4n– LN464 shows three chromosome 12s with both red and green hybridization signals, indicating the loss of one copy of the Smac locus. M to O, dual hybridization with BAC clone (RPCI11-367I6) probe for caspase-3 (green) and control probe CEP 4 (red) was done on three glioma cell lines. M, metaphase cell of the 4n– LN464 shows only having three chromosome 4s with both caspase-3 (green) and control (red), indicating the loss of one copy of the caspase-3 locus in this cell. N, metaphase cell of 3n– U373MG cell line shows two chromosome 4 both having red and green signals and a derivative chromosome 4 lacking the green hybridization signal, indicating the cell losing a caspase-3 locus. O, 3n– LN215 shows only having two chromosome 4s each with both green and red hybridization signals. However, a derivative chromosome 18 with a gain of the caspase-3 locus (green signal) was also observed.

 
As for the caspase-8 locus at 2q33-q34, the majority (92%) of 3n– TRAIL/chemotherapy-resistant LN215 showed loss of a copy of the region because of the loss of a chromosome 2; thus, only two positive FISH signals in the 3n– cells were observed with the 2q33-q34 region probe (Fig. 4D). However, 75% cells of TRAIL/chemotherapy-resistant U373MG showed no loss of chromosome 2. In TRAIL-resistant cell lines, loss of the caspase-8 region (2q33-q34) was found in 63% of LN443 cells examined due to loss of a chromosome 2; thus, 3n cells of this line were found only having two FISH signals with the 2q33-q34 region probe (Fig. 4E). No loss of caspase-8 region was determined in U138MG, and only 38% of U118MG cells analyzed showed the loss of this region. The 4n- TRAIL-resistant cell line LN464 had 4 copies of chromosome 2 each containing a caspase-8 locus in the majority of the cells examined, but some of the cells showed a dicentric chromosome, dic(2;19)(q31;q12) lacking the caspase-8 signal (Fig. 4F). The majority of cells examined from 4n– TRAIL-sensitive line LN71 showed no loss of the caspase-8 region; thus, no loss of this locus was assigned.

The two TRAIL/chemotherapy-resistant lines (LN215 and U373MG) showed loss of a copy of the Bid locus at 22q11.2 in 77% and 95% cells analyzed, respectively; thus, only two copies of Bid signal were observed in the majority of cells examined from these 3n– cell lines (Fig. 4G and H). No net loss of the Bid region was found in the TRAIL-sensitive cell line LN71. Most TRAIL-resistant cell lines studied also had no loss or gain of the Bid locus (Fig. 4I). Similarly, loss of a copy of the Smac locus at 12q24.3 was found in the two TRAIL/chemotherapy-resistant lines LN215 and U373MG, as 92% and 80% of the cells from these two lines lost a copy of chromosome 12, which contained the Smac locus. This was confirmed by FISH study (Fig. 4J and K). Again, no loss of such gene copy was found in the majority of TRAIL-sensitive LN71 cells examined. Loss of a copy or copies of Smac locus were commonly found in TRAIL-resistant cell lines (4L) with exception of the U138MG.

As for the other three caspase loci studied, no loss of caspase-9 region 1p36.1-p36.3 was observed in the two TRAIL/chemotherapy-resistant cell lines. However, loss, no loss, and gain of the region were found in TRAIL-resistant cells of LN464, U138MG, and LN443, respectively. Almost equal numbers of cells examined in the TRAIL-sensitive line LN71 showed loss or no loss of the region, which contained the caspase-9 gene. Loss of gene copy regarding the caspase-3 locus at 4q35 was commonly observed in all TRAIL-resistant cell lines (Fig. 4M) and in the TRAIL-sensitive cell line studied. On the other hand, only one TRAIL/chemotherapy-resistant line U373MG showed 90% of the cells losing a copy of this locus (Fig. 4N). The other TRAIL/chemotherapy-resistant line LN215 lost a chromosome 4 but was compensated by derivative chromosome, der(18)t(4;18)(q21;q21) with a gain of the caspase-3 locus (Fig. 4O). All TRAIL-resistant lines and the TRAIL-sensitive line showed loss of the region 10q25.1-q25.2 containing the caspase-7 locus, whereas loss of this locus was found in only one TRAIL/chemotherapy-resistant line (LN215).

Genomic aberrations in human malignant gliomas. To investigate whether the genomic aberrations in TRAIL apoptotic genes identified in malignant glioma cell lines can also be observed in human malignant gliomas, FISH studies with DNA probes that detect DR5, caspase-8, BID, and Smac gene loci were conducted on two primary cultures (ED189BT and ED326BT) derived from malignant glioma tumors surgically removed from patients (7). ED189BT is hyperdiploid (2n+) with chromosome number 49 to 57 based on 20 metaphase spreads examined, whereas ED326BT is near tetraploid (4n) with a chromosome number of 80 to 116 based on 17 metaphases analyzed. Loss of a chromosome 2 that contains the caspase-8 locus was found in 20% of the 2n+ ED189BT culture (Fig. 5A ) based on 100 cells (metaphase and interphase cells) examined. No loss of the other three gene loci (DR5, Bid, and Smac) was observed in this culture (Fig. 5B-D). Loss of gene copy regarding these four gene loci was not detected in ED326BT primary cultures. Four red signals of Smac loci (Fig. 5E) and four green signals of the caspase-8 loci (Fig. 5F) were observed in most of the cells examined. Similarly, the majority of cells analyzed from this culture showed at least four green DR5 locus signals and four green Bid locus signals (data no shown). To correlate the genomic aberration in caspase-8 locus with the sensitivity of primary cultures, we treated each of the cultures with TRAIL, alone or in combination with cisplatin. The results show that ED326BT was sensitive to TRAIL treatment (Fig. 5G), whereas ED189BT was TRAIL resistant (Fig. 5H). Treatment with cisplatin significantly enhanced TRAIL killing of ED189BT; however, a portion of the cells remained resistant to the combination treatment of TRAIL and cisplatin (Fig. 5H). The identification of the loss of caspase-8 locus in 20% ED189BT suggests that this genomic alteration may in part contribute to the resistance of ED189BT to the combination treatment of TRAIL and cisplatin.


Figure 5
View larger version (32K):
[in this window]
[in a new window]
  		Fig. 5. Genomic aberrations and TRAIL sensitivity in human malignant glioma cultures. A to F, FISH analysis with DNA probes detecting DR5, caspase-8, Bid, and Smac loci in primary culture ED189BT. Four different metaphase spreads from the culture were hybridized with caspase-8, DR5, Smac, and Bid, respectively. A, only one caspase-8 signal (green fluorescent and arrow) was observed in a chromosome 2 (control probe CEP2 shows red fluorescent). An interphase nuclear (inset) shows one green hybridization signal of the caspase-8 locus (arrowhead). B, two chromosome 8s identified with 8q11.1 BAC clone probe RP11-269N2 (red signals) each shows positive DR5 signals (green signal and arrows). C, two chromosome 12s identified with CEP112 (green signals) each shows positive Smac signals (red signals and arrows). D, two chromosome 22s identified with 22q11.1 clone P109L3 probe (red signal) each shows positive Bid signals (green signal and arrows). E, metaphase cell form ED326BT shows four chromosome 12 (identified by CEP12, green signals) each having a positive hybridization signal with DNA probe detecting Smac locus (red signal and arrow). An interphase nuclear (inset) shows four hybridization signals of the Smac locus (red signal and arrowhead). F, a metaphase cell form ED326BT shows four chromosome 2s (identified by CEP2, red signals) each having a positive hybridization signal with DNA probe detecting the caspase-8 locus (green signal and arrow). G, cell death analysis of ED326BT primary culture after 16 hours of treatment with TRAIL at the concentrations indicated. H, effects of chemotherapy on TRAIL sensitivity in the primary culture ED189BT. The cell culture was treated with 300 ng/mL TRAIL in the combination with cisplatin (Cis) or temozolomide (TMZ) at the concentrations indicated and then subjected to cell death analysis. Points, mean U (n = 6); bars, SE.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We have examined a large panel of malignant glioma cell lines and identified TRAIL-sensitive and TRAIL-resistant cell lines (5, 7). TRAIL induces apoptosis in the sensitive cell lines through caspase-8-initiated extrinsic and intrinsic pathways (7). Caspase-8-initiated pathways, however, are inhibited in the resistant cell lines due to the recruitment of cellular Fas associate death domain-like IL-1ß-converting enzyme-inhibitory protein (c-FLIP) and phosphoprotein enriched in astrocytes 15 kDa/(PEA-15)/phosphoprotein enriched in diabetes (PEA-15/PED) to the death-inducing signaling complex where they inhibit caspase-8 cleavage (46). Conventional chemotherapy agents, such as camptothecin, cisplatin, and etoposide, down-regulates c-FLIP and PED expression and thus sensitizes the resistant glioma cells to TRAIL-induced apoptosis (7). This combination treatment may prove to be effective in clinical treatment of malignant gliomas. In this study, however, we have identified two malignant glioma cell lines that are resistant to the combination treatment with TRAIL and chemotherapeutic agents (cisplatin, doxorubicin, etoposide, and temozolomide). Using a systematic approach, we have further shown the simultaneous loss of chromosomal regions containing DR4/DR5, caspase-8, Bid, and Smac loci with a concomitant reduction in the expression of the corresponding TRAIL apoptotic proteins in these cell lines. Furthermore, we have examined two primary cultures derived form patients' gliomas and identified loss of a chromosome that harbors the caspase-8 locus in one of the cultures. To our knowledge, the study presented here is the first attempt to correlate the functional phenotype of human malignant glioma cell lines and primary cultures to their genomic defects in TRAIL apoptotic genes.

CGH has been used to identify chromosomal abnormalities in malignant gliomas (23–25). In this study, we began genomic analysis of malignant glioma cell lines with CGH to assess the overall genomic imbalance and identified genomic imbalances in the chromosomal regions that harbor DR4/DR5, FADD, caspase-3, caspase-7, caspase-8, caspase-9, Bid, and Smac genes. Loss of these chromosomal regions in U373MG is in accord with CGH studies from other laboratories (47, 48). The present CGH study has further revealed that the multiple losses (three or more) of these regions occurred in four of six TRAIL-resistant cell lines and are more common in TRAIL- and chemotherapy-resistant cell lines (Table 1). Subsequently, loss, no loss, or gain of gene copy estimation by a combination of G-banding/spectral karyotyping and FISH analysis confirmed the CGH findings in all eight key regions examined in the TRAIL/chemotherapy-resistant cell lines (Table 1). This finding further validated that CGH could detect loss of a copy of the specific chromosome region in triploid cells. The gene copy estimation results are also in agreement with the CGH findings in the majority of cases in the two tetraploid cell lines examined (Table 3). This suggests to a certain extend that CGH could detect loss or gain of one to two copies of a particular region in the near 4n tumor cell line as well. It is interesting to note that loss of a region, 1p36.1-p36.3 (caspase-9), detected by CGH in the tetraploid TRAIL-sensitive cell line LN71 was observed in only 39% of cells by G-banding/spectral karyotyping and FISH. On the other hand, in only half of the chromosomal regions examined did CGH findings fit well with the gene copy estimation in the pentaploid cell line U118MG and the 3n/6n cell line LN443. This is perhaps either because that the percentage of cells with such aberration is too low (49) or CGH may not be able to detect loss or gain of a single copy of the chromosomal region in pentaploidy or hexaploidy (Table 1).

One of the salient features in the present study is the loss of a copy in each of four chromosomal regions that harbor DR4/DR5, caspase-8, Bid, and Smac loci, which corresponded well with the low expression profile of the corresponding proteins in two 3n– glioma cell lines that are resistant to the TRAIL and chemotherapy treatment. These findings could provide a mechanism for the ability of the cells to resist the combination treatment. However, the 3n TRAIL-resistant cell line U138MG and the 4n TRAIL-sensitive line LN71 were found to have lost a copy of the DR5 region in 68% and 83% cells examined, respectively, with no apparent effect on their DR5 protein expression. It remains to be investigated how polyploidy affects the gene expression and defines the epigenetic phenotypes of the cells. Several possible mechanisms have been suggested and include intergenome function of chromatin remodeling factors affecting DNA methylation modification, chromatin packaging, and gene mutation and deletions (50). Indeed, DR4 and DR5 mutations have been reported in lung, head, neck, and breast cancers (51, 52), and lack of caspase-8 expression in neuroblastoma cells due to methylation has been shown in neuroblastoma (53).

FISH has proven to be very useful for gene copy estimation and determination of whether a structural aberrant chromosome has indeed lost, retained, or gained a particular gene locus of interest. For instance, in the TRAIL-resistant cell line LN464, a chromosome 8 with an inversion [inv(8)(p11.2p21)] was identified by G-banding/spectral karyotyping analysis. The DR5 gene was indeed found to be relocated (no loss) close to the centromeric region by FISH (Fig. 4C). Such relocation of the DR5 locus in this case seemed to have no effect on the expression of the DR5 gene in this cell line (Fig. 1C).

All the seven human malignant glioma cell lines examined show the near polyploidy from 3n– to 6n– chromosomes numbers, with complex numerical and structural aberrations, resulting in various phenotypes in expression of TRAIL apoptotic genes. This study provides a systematic approach to detect these genomic defects in glioma cells in correlation to the cell sensitivity to TRAIL treatment; however, further study is needed to investigate how these chromosomal aberrations affect the expression of the genes in the regions, thus contributing to the phenotypes of malignant gliomas in their responsiveness to TRAIL-induced apoptosis. Recombinant human TRAIL can kill many types of cancer cells but spares normal cell in cultures and experimental animals (54, 55); thus, it is currently under clinical development as a cancer therapeutic agent. In this study, however, we have identified established malignant glioma cell lines that are resistant to the TRAIL and chemotherapy treatment in correlation with the genomic loss of key TRAIL apoptotic genes. Moreover, we have identified the genomic loss of a caspase-8 locus in a portion of primary culture cells derived from a human malignant glioma tumor. This primary glioma tumor cell culture has a hyperdiploid karyotype and loss of a copy of the caspase-8 gene seemed correlated well with its resistance to the combined TRIAL and chemotherapy treatment. These genomic defects could be therefore important for predicting the responsiveness of malignant gliomas to the treatment with TRAIL and chemotherapy drugs in future clinical trials.


   Acknowledgments
 
We thank Dr Wafik S. El-Deiry (University of Pennsylvania, Philadelphia, PA) for kindly providing the DR5 cNDA clone, Penny Coates and Shirley Lammers for their technical assistance, and Dr. Shannon Matheny for critically reading the article.


   Footnotes
 
Grant support: Taiwan National Health Research Institute grant NHRI-EX92-9207SI (C-C. Lin), Taiwan National Sciences Council grants NSC90-2745-P040-048 and NSC92-2320-B-040-048 (Y-C. Li), National Cancer Institute of Canada (C. Hao), Southeastern Brain Tumor Foundation of the United States (C. Hao), and NIH grant CA86335 (E.G. Van Meir).

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.

Received 9/ 9/05; revised 1/27/06; accepted 3/ 2/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Legler JM, Ries LA, Smith MA, et al. Cancer surveillance series [corrected]: brain and other central nervous system cancers: recent trends in incidence and mortality. J Natl Cancer Inst 1999;91:1382–90.[Abstract/Free Full Text]
  2. Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82.[CrossRef][Medline]
  3. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 1996;271:12687–90.[Abstract/Free Full Text]
  4. Nagane M, Pan G, Weddle JJ, Dixit VM, Cavenee WK, Huang HJ. Increased death receptor 5 expression by chemotherapeutic agents in human gliomas causes synergistic cytotoxicity with tumor necrosis factor-related apoptosis-inducing ligand in vitro and in vivo. Cancer Res 2000;60:847–53.[Abstract/Free Full Text]
  5. Hao C, Beguinot F, Condorelli G, et al. Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis in human malignant glioma cells. Cancer Res 2001;61:1162–70.[Abstract/Free Full Text]
  6. Ehtesham M, Kabos P, Gutierrez MA, et al. Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2002;62:7170–4.[Abstract/Free Full Text]
  7. Song JH, Song DK, Pyrzynska B, Petruk KC, Van Meir EG, Hao C. TRAIL triggers apoptosis in malignant glioma cells through extrinsic and intrinsic pathways. Brain Pathol 2003;13:539–53.[Medline]
  8. Knight MJ, Riffkin CD, Muscat AM, Ashley DM, Hawkins CJ. Analysis of FasL and TRAIL induced apoptosis pathways in glioma cells. Oncogene 2001;20:5789–98.[CrossRef][Medline]
  9. Rohn TA, Wagenknecht B, Roth W, et al. CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced cytochrome c release. Oncogene 2001;20:4128–37.[CrossRef][Medline]
  10. Saito R, Bringas JR, Panner A, et al. Convection-enhanced delivery of tumor necrosis factor-related apoptosis-inducing ligand with systemic administration of temozolomide prolongs survival in an intracranial glioblastoma xenograft model. Cancer Res 2004;64:6858–62.[Abstract/Free Full Text]
  11. Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2002;2:420–30.[CrossRef][Medline]
  12. Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 1995;81:505–12.[CrossRef][Medline]
  13. Bodmer JL, Holler N, Reynard S, et al. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol 2000;2:241–3.[CrossRef][Medline]
  14. Kischkel FC, Hellbardt S, Behrmann I, et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J 1995;14:5579–88.[Medline]
  15. Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 1996;85:817–27.[CrossRef][Medline]
  16. Wagenknecht B, Glaser T, Naumann U, et al. Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma. Cell Death Differ 1999;6:370–6.[CrossRef][Medline]
  17. Deng Y, Lin Y, Wu X. TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev 2002;16:33–45.[Abstract/Free Full Text]
  18. Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 1998;94:491–501.[CrossRef][Medline]
  19. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000;102:33–42.[CrossRef][Medline]
  20. Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000;102:43–53.[CrossRef][Medline]
  21. Kirsch DG, Doseff A, Chau BN, et al. Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c. J Biol Chem 1999;274:21155–61.[Abstract/Free Full Text]
  22. Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 1997;89:175–84.[CrossRef][Medline]
  23. Kim DH, Mohapatra G, Bollen A, Waldman FM, Feuerstein BG. Chromosomal abnormalities in glioblastoma multiforme tumors and glioma cell lines detected by comparative genomic hybridization. Int J Cancer 1995;60:812–9.[Medline]
  24. Mohapatra G, Moore DH, Kim DH, et al. Analyses of brain tumor cell lines confirm a simple model of relationships among fluorescence in situ hybridization, DNA index, and comparative genomic hybridization. Genes Chromosomes Cancer 1997;20:311–9.[CrossRef][Medline]
  25. Squire JA, Arab S, Marrano P, et al. Molecular cytogenetic analysis of glial tumors using spectral karyotyping and comparative genomic hybridization. Mol Diagn 2001;6:93–108.[CrossRef][Medline]
  26. Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 1992;258:818–21.[Abstract/Free Full Text]
  27. Schrock E, du Manoir S, Veldman T, et al. Multicolor spectral karyotyping of human chromosomes. Science 1996;273:494–7.[Abstract]
  28. MacFarlane M, Ahmad M, Srinivasula SM, Fernandes-Alnemri T, Cohen GM, Alnemri ES. Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J Biol Chem 1997;272:25417–20.[Abstract/Free Full Text]
  29. Pan G, O'Rourke K, Chinnaiyan AM, et al. The receptor for the cytotoxic ligand TRAIL. Science 1997;276:111–3.[Abstract/Free Full Text]
  30. Kim PK, Dutra AS, Chandrasekharappa SC, Puck JM. Genomic structure and mapping of human FADD, an intracellular mediator of lymphocyte apoptosis. J Immunol 1996;157:5461–6.[Abstract]
  31. Kischkel FC, Kioschis P, Weitz S, Poustka A, Lichter P, Krammer PH. Assignment of CASP8 to human chromosome band 2q33->q34 and Casp8 to the murine syntenic region on chromosome 1B-proximal C by in situ hybridization. Cytogenet Cell Genet 1998;82:95–6.[CrossRef][Medline]
  32. Tiso N, Pallavicini A, Muraro T, et al. Chromosomal localization of the human genes, CPP32, Mch2, Mch3, and Ich-1, involved in cellular apoptosis. Biochem Biophys Res Commun 1996;225:983–9.[CrossRef][Medline]
  33. Hadano S, Nasir J, Nichol K, et al. Genomic organization of the human caspase-9 gene on Chromosome 1p36. 1-p36.3. Mamm Genome 1999;10:757–60.[Medline]
  34. Footz TK, Birren B, Minoshima S, et al. The gene for death agonist BID maps to the region of human 22q11.2 duplicated in cat eye syndrome chromosomes and to mouse chromosome 6. Genomics 1998;51:472–5.[CrossRef][Medline]
  35. Ishii N, Maier D, Merlo A, et al. Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol 1999;9:469–79.[Medline]
  36. Parney IF, Petruk KC, Zhang C, Farr-Jones M, Sykes DB, Chang LJ. Granulocyte-macrophage colony-stimulating factor and B7–2 combination immunogene therapy in an allogeneic Hu-PBL-SCID/beige mouse-human glioblastoma multiforme model. Hum Gene Ther 1997;8:1073–85.[Medline]
  37. Bryndorf T, Kirchhoff M, Rose H, et al. Comparative genomic hybridization in clinical cytogenetics. Am J Hum Genet 1995;57:1211–20.[Medline]
  38. Kirchhoff M, Gerdes T, Rose H, Maahr J, Ottesen AM, Lundsteen C. Detection of chromosomal gains and losses in comparative genomic hybridization analysis based on standard reference intervals. Cytometry 1998;31:163–73.[CrossRef][Medline]
  39. Wu GS, Burns TF, McDonald ER, et al. KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat Genet 1997;17:141–3.[CrossRef][Medline]
  40. Lin CC, Sasi R, Lee C, Fan YS, Court D. Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8. Chromosoma 1993;102:333–9.[CrossRef][Medline]
  41. Footz TK, Brinkman-Mills P, Banting GS, et al. Analysis of the cat eye syndrome critical region in humans and the region of conserved synteny in mice: a search for candidate genes at or near the human chromosome 22 pericentromere. Genome Res 2001;11:1053–70.[Abstract/Free Full Text]
  42. Lin CC, Sasi R, Fan YS, Chen ZQ. New evidence for tandem chromosome fusions in the karyotypic evolution of Asian muntjacs. Chromosoma 1991;101:19–24.[CrossRef][Medline]
  43. Apte SS, Mattei MG, Olsen BR. Mapping of the human BAX gene to chromosome 19q13.3-q13.4 and isolation of a novel alternatively spliced transcript, BAX delta. Genomics 1995;26:592–4.[CrossRef][Medline]
  44. Herberg JA, Phillips S, Beck S, et al. Genomic structure and domain organisation of the human Bak gene. Gene 1998;211:87–94.[CrossRef][Medline]
  45. Veldman T, Vignon C, Schrock E, Rowley JD, Ried T. Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping. Nat Genet 1997;15:406–10.[CrossRef][Medline]
  46. Xiao C, Yang BF, Asadi N, Beguinot F, Hao C. Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells. J Biol Chem 2002;277:25020–5.[Abstract/Free Full Text]
  47. Kubota H, Nishizaki T, Harada K, et al. Identification of recurrent chromosomal rearrangements and the unique relationship between low-level amplification and translocation in glioblastoma. Genes Chromosomes Cancer 2001;31:125–33.[Medline]
  48. Weber RG, Rieger J, Naumann U, Lichter P, Weller M. Chromosomal imbalances associated with response to chemotherapy and cytotoxic cytokines in human malignant glioma cell lines. Int J Cancer 2001;91:213–8.[Medline]
  49. Kallioniemi OP, Kallioniemi A, Piper J, et al. Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosomes Cancer 1994;10:231–43.[Medline]
  50. Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:1074–80.[Abstract/Free Full Text]
  51. Fisher MJ, Virmani AK, Wu L, et al. Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer. Clin Cancer Res 2001;7:1688–97.[Abstract/Free Full Text]
  52. Shin MS, Kim HS, Lee SH, et al. Mutations of tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and receptor 2 (TRAIL-R2) genes in metastatic breast cancers. Cancer Res 2001;61:4942–6.[Abstract/Free Full Text]
  53. Hopkins-Donaldson S, Bodmer JL, Bourloud KB, Brognara CB, Tschopp J, Gross N. Loss of caspase-8 expression in highly malignant human neuroblastoma cells correlates with resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Cancer Res 2000;60:4315–9.[Abstract/Free Full Text]
  54. Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 1999;104:155–62.[Medline]
  55. Hao C, Song JH, Hsi B, et al. TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice. Cancer Res 2004;64:8502–6.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Y.-C.
Right arrow Articles by Lin, C.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Y.-C.
Right arrow Articles by Lin, C.-C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal