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Cell, Tumor, and Stem Cell Biology |
1 Department of Biomedicine, University of Bergen; 2 The Gade Institute, University of Bergen, Haukeland University Hospital, Bergen, Norway; and 3 NorLux Neuro-Oncology, Centre Recherche Public de la Santé, Luxembourg
Requests for reprints: Dorota Goplen, The Gade Institute, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. Phone: 475-597-2616; E-mail: dorota.goplen{at}biomed.uib.no.
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Abstract |
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Introduction |
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Another methodologic problem is that xenotransplanting human glioma cell lines into the central nervous system (CNS) produces circumscribed tumors with little or no single cell infiltration into the brain parenchyma (1). To avoid this problem, we have developed a xenograft model where human brain tumor biopsy spheroids are transplanted into the nude rat brain. The tumors derived from such spheroids show a highly infiltrative behavior in the CNS, which reflects the invasive characteristics of the human tumors in situ (low-generation tumors; refs. 8, 9). On serial transplantation in vivo, the tumors will develop a less invasive and more angiogenic phenotype (high-generation tumors). Thus, in vivo tumor xenografts obtained from the same patient provide two different phenotypes: one that is predominantly invasive, showing little or no signs of angiogenesis, and one that is less invasive and predominantly angiogenic. By comparing the two different phenotypes using a proteomics approach, including the use of two-dimensional protein electrophoresis (2DE), mass spectrometry (MS) and bioinformatics, we identified novel proteins that were differentially expressed by the invasive phenotype. Using both in vitro and in vivo assay systems, we present one of the identified proteins, protein disulfide isomerase (PDI) that was overexpressed in the invasive phenotype. Moreover, its involvement in glioma cell invasion was confirmed in functional assays, where PDI inhibition caused a reduced cell migration and invasion. Finally, we show that PDI is strongly expressed in human glioblastoma multiforme biopsies taken from the invasive rim of infiltrative brain tumor.
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Materials and Methods |
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Spheroids were prepared as described previously (10). Briefly, tissue samples were minced into 0.5-mm fragments, placed into 80-cm2 tissue culture flasks (Nunc, Roskilde, Denmark), and base coated with 10 mL of 0.75% agar (Difco, Detroit, MI) in culture medium (DMEM). The spheroids were maintained in a standard tissue culture incubator with 5% CO2 in air and 100% relative humidity at 37°C and the medium was changed once weekly. After 1 to 2 weeks in culture, spheroids with diameters between 200 and 300 µm were selected for i.c. implantation (Fig. 1 ).
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Animal experiments. Nude rats (Han:rnu/rnu Rowett) were fed a standard pellet diet and provided water ad libitum. All procedures were approved by The National Animal Research Authority. Biopsy spheroids were stereotactically implanted into the right brain hemisphere as described elsewhere (8). The animals were sacrificed when symptoms developed, and the brains were then removed. All surgical procedures were done on animals anesthetized by s.c. injection of Dormicum (Roche, Basel, Switzerland), 0.2 g/100 g of body weight, and fentanyl/fluanisone (Hypnorm, Janssen, Oxford, United Kingdom; fentanyl citrate, 0.15 mg/mL; fluanisone, 10 mg/mL), 0.0126 g/100 g of body weight. The rats were immobilized in a stereotactic frame (model 900, David Kopf Instruments, Tujunga, CA). After local anesthesia with Xylocain (AstraZeneca, Zug, Switzerland), a burr hole was made 3.0 mm to the right of the sagittal suture and 1 mm posterior to the bregma. PBS (25 µL) containing 10 spheroids was injected into the forebrain to a depth of 2.5 mm under the brain surface. Closure was effected with 3.0 ethilon suture. Tumor growth was monitored at regular intervals during 4 to 20 weeks after implantation using a MRI Magnetom Vision Plus 1.5 T scanner (Siemens, Erlangen, Germany) and a small loop finger coil as described previously (12, 13). The animals were sacrificed by CO2 inhalation when neurologic signs were evident. The tumors were removed and new spheroids were generated, which again were transplanted into new animals. A schematic description of the experimental procedure is provided in Fig. 1. The brains were removed and fixed in 4% formaldehyde or snap frozen in liquid N2 for further studies.
Two-dimensional protein electrophoresis. Novel proteins were identified by 2DE followed by gel comparisons. Differentially expressed proteins were selected manually, excised, and trypsinized.
The tryptic digest was applied on a MALDI-TOF plate and peptide mass spectra were generated. The achieved mass spectra were compared with in silico–generated theoretical ones and the score, indicating the probability of correct protein identification was obtained. Only proteins identified with significant scores were further verified.
The brain tumor specimens were stored in liquid N2. After thawing, they were washed in TRIS/sucrose solution [0.25 mol/L sucrose in 10 mmol/L Tris (pH 7.4); Tris, Merck, Darmstadt, Germany; sucrose, Sigma-Aldrich, St. Louis, MO] and placed in sample buffer containing 7 mol/L urea, 2 mol/L thiourea (Merck), 4% CHAPS (Sigma-Aldrich), 100 mmol/L DTT (Merck), and 1% pharmalyte (Amersham Biosciences, Uppsala, Sweden) pI 3-10: pI 5-6 = 3:1. Afterwards, the tissue samples were homogenized 3 x 10 seconds, sonicated 3 x 1 seconds, and centrifuged at 1,600 x g for 20 minutes at 4°C using an Eppendorf centrifuge (Eppendorf, Hamburg, Germany). The supernatant was transferred to a new tube and the protein concentration was estimated using the Bradford reagent (Bio-Rad, Hercules, CA). For the analytic gels, a protein load of 100 µg per gel was applied. The protein load for micropreparative gels was 400 µg/gel.
The first dimension of separation was carried out on IPGphore (Amersham Biosciences) at 20°C. The desired protein amount, 100 µg for analytic gels and 400 µg for the micropreparative ones, was diluted in 350 µL sample buffer and applied on an 18-cm (pH 3-10) linear immobilized pH gradient (IPG) Immobiline DryStrip IPG (Amersham Biosciences) by in-sample rehydration. After 1 hour of passive rehydration, the strips were covered with mineral oil (Bio-Rad) to prevent drying, and 50 V voltage was applied overnight (16 hours). The day after, the electrodes were covered with Milli-Q water moistened paper pads and the voltage increased to 250 V for 15 minutes followed by a gradient to 8,000 V and step-in-hold 8,000 V to final 60,000 Vhrs. After 1 hour of separation, the electrode pads were changed to remove the electrolytes. After the first dimension of separation, the IPG strips were stored at –80°C until they were subjected to the second dimension of separation.
Before the second dimension of electrophoresis, the strips were equilibrated for 15 minutes in solution containing 100 mmol/L DTT, 6.5 mol/L urea (Merck), 26% (w/v) glycerol (Merck), 2% (w/v) SDS solution (Bio-Rad), and 50 mmol/L Tris-HCl (pH 6.8). Thereafter, they were subjected to a new equilibration solution for 15 minutes, where DTT was replaced by 243 mmol/L iodoacetamide (Sigma-Aldrich). After equilibration, the strips were applied on 13.75% acrylamide/N,N'-methylenebisacrylamide (Bio-Rad) gel, and the electrophoresis was carried out overnight. Separation in the second dimension of electrophoresis was carried out on a Protean II Cell (Bio-Rad) using 13.75% SDS-polyacrylamide gel with 5% stacking gel at a temperature of 15°C and an initial voltage of 35 V for 7.5 hours, 65 V for 8 hours, and 95 V until the electrophoresis was finished.
After the second dimension electrophoresis, the analytic gels were silver stained, dried, and analyzed manually. Spots overexpressed in the invasive phenotype in both patients were selected. Thereafter, micropreparative gels with higher protein load were prepared and stained with SYPRO Ruby (Bio-Rad) immediately after the SDS-PAGE electrophoresis according to the manufacturer's protocol. The gels were incubated in the SYPRO Ruby overnight. The images of SYPRO Ruby–stained gels were obtained on image analyzer LAS-100 (Fuji, Tokyo, Japan).
Mass spectrometry. After manual excision, the differentially expressed spots were stored in -80°C until further analysis. During the preparation of protein samples for MS, the gel pieces were washed in NH4HCO3/acetonitrile (Sigma-Aldrich) solution and dried in speed-vac for 20 minutes and in-gel trypsin digestion (Promega, Madison,WI) was done overnight at 37°C. Thereafter, the peptides were extracted, lyophilized, reconstituted, and mixed with 
-cyano-4-hydroxycinnamic acid (Promega) matrix solution directly on the MALDI target plate. Peptide mass spectra were generated on an Ultraflex MALDI-TOF (Bruker Daltonics, Bremen, Germany). The experimental peptide mass spectrum was matched to the theoretical spectra using a peptide mass fingerprinting technique and a MASCOT search engine (14). A probability-based scoring, showing a match between the experimental data and mass values calculated from candidate peptide sequences, was then obtained.
Western blot. The tissue pieces were homogenized in homogenization buffer, containing CHAPS, K2HPO4, KH2PO4, EDTA (Merck), and protease inhibitor cocktail (Complete, Roche Diagnostics, Manheim, Germany) and centrifuged and the supernatant was transferred into a new tube before the protein concentration was estimated. The samples were kept frozen at -80°C until use.
A 12.5% gel was prepared and 30 µg proteins were subjected to SDS-PAGE electrophoresis at 100 V for 2 hours. The bands were transferred onto a polyvinylidene difluoride transfer membrane Hybond-P (Amersham Biosciences). Expression of PDI was detected using an anti-PDI mouse monoclonal antibody (Alexis Biochemicals, Lausen, Switzerland) at a concentration of 1:1,000. The images were obtained on image analyzer LAS-100.
Immunohistochemistry. Monoclonal mouse antibodies to PDI were applied on formalin-fixed, paraffin-embedded tissue slices at a concentration of 1:100 and 1:200. The application of a secondary antibody and visualization was done with the DakoCytomation EnVision+ System-HRP kit according to the protocol of the manufacturer (DakoCytomation, Carpinteria, CA).
For immunofluorescence, the cells were fixed with paraformaldehyde in PBS, permeabilized with Triton X-100 (Sigma) in PBS, and incubated with primary PDI antibody at a concentration of 1:100. The secondary antibody used was isotope-specific goat anti-mouse conjugated to FITC (SouthernBiotech, Birmingham, AL).
Flow cytometry. U373 cells were grown as monolayers to 90% confluence in 80-cm2 cell culture flasks. Thereafter, the cells were dissociated with 2 mmol/L EDTA solution without trypsin. The cell suspension was then transferred to 15 mL centrifuge tubes and the cell numbers were determined using a Bürker chamber. Thereafter, the cell suspensions were centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, and the pelleted cells were resuspended in fresh 4% paraformaldehyde in PBS. After fixation, 4 x 105 cells were transferred into 96-well multidishes (V-shaped bottom). The dishes were centrifuged at 1,000 rpm for 5 minutes. The supernatant was discarded and the cells were washed twice in PBS. The cells were not permeabilized. The cells were then incubated with a mouse monoclonal antibody to PDI at a dilution of 1:50 for 45 minutes on ice. After washing twice in PBS, the cells were incubated for 45 minutes with a FITC-conjugated goat anti-mouse F(ab')2 secondary antibody (DAKO Denmark A/S, Glostrup, Denmark). All the steps were carried out on ice. The control cells were incubated with the secondary but not with the primary antibody. After incubation, the cells were washed again in PBS and stored at 4°C until analysis. The flow cytometry was done using a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA). To verify PDI expression at the cell surface, nonpermeabilized cells were also examined by confocal microscopy. The same cells, as used for the flow cytometric analysis, were mounted on slides using 4',6-diamidino-2-phenylindole (DAPI) containing mounting medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA) whereupon they were analyzed using a Zeiss confocal microscope (Zeiss LSM 510 Meta, Jena, Germany).
Adhesion assay. The cells were grown in 80-cm2 culture flasks and nonenzymatically detached from the bottom as described before. Thereafter, they were centrifuged at 800 rpm for 5 minutes and the pellet was resuspended in 5 mL medium without serum. The cells were then counted and incubated for 1 hour on ice with a monoclonal PDI antibody diluted in complete medium without serum. The control cells were incubated in complete medium without serum. After 1 hour, the cells were counted and equal number of control and treated cells was seeded in 5 parallel wells of 24- and 48-well multidishes. The number of attached cells was determined after 2 and 24 hours. The experiment was repeated four times with different number of seeded cells.
Migration assay. To assess functional variables related to PDI expression, six spheroids derived from the highly invasive rat xenograft (low-generation tumor) and six spheroids derived from the less invasive, angiogenic xenograft (high-generation tumor) were placed in 24-well plates, one in each well and overlaid with 0.5 mL medium. After 4 hours, 0.5 mL medium containing 10 mmol/L bacitracin (Sigma), which is a specific inhibitor of PDI (15, 16), was added to three wells containing low-generation and three containing high-generation spheroids to a final concentration 5 mmol/L bacitracin. The remaining six wells (three with low-generation spheroids and three with high-generation spheroids, respectively) represented the control group, which received normal growth medium. The experiments were done twice.
In one set of experiments, bacitracin was withdrawn from the cell cultures (treatment day 0-2, treatment day 0-4, and treatment day 0-7). There were 6 spheroids in each treatment group (total of 24 spheroids).
To verify that the migration inhibition seen by bacitracin represented a PDI-specific effect, we also did the same experiment using a PDI-specific antibody that inhibited the expression of PDI on U373 cells (which express high levels of PDI). There were three U373 spheroids in each treatment group [i.e., treatment with 2.5 and 5 mmol/L bacitracin, treatment with PDI antibody at the dilution of 1:10, 1:50, 1:100, and 1:500, and 6 spheroids in the control group (total of 24 spheroids)]. The experiments were done twice and terminated after 4 days.
The diameter of the spheroids was measured at the time of plating, and the cell migration area was measured daily using an inverted phase-contrast light microscope with a micrometer in the ocular. Cell migration was calculated as the distance from the center of the spheroid to the population of migrating cells most distant from the spheroid minus the original spheroid radius. The medium was changed every second day. The longest treatment with bacitracin was 7 days. Measurements were recorded for up to 13 days. The monoclonal PDI antibody was diluted in medium and added to the culture in the same way as for bacitracin. In the dose response studies, the following bacitracin concentrations were used: 2.5, 5, and 10 mmol/L. The experiments with monoclonal antibody to PDI were carried out in 96-well plates containing 160 µL medium.
In the experiments where possible additive effects of bacitracin and ß1 and ß3 integrin blocking antibodies on glioma cell migration were tested, the U373 glioma cell line spheroids were placed in 48-well multidishes, one in each, and covered with 200 µL medium. After 4 hours, when the spheroids were attached to the bottom, the normal medium was replaced with medium containing bacitracin at concentrations of 2.5 and 5 mmol/L with or without ß1 integrin blocking antibody (dilution 1:10, 1:50, and 1:100; SouthernBiotech). The same experiment was also done using a ß3 blocking antibody (Chemicon International, Temecula, CA). Cell migration was then measured at regular intervals as decribed above.
Invasion assay. The collagen invasion assay was carried out as described previously (17). Briefly, the collagen solution was prepared by mixing 3.2 mg/mL collagen type I in 0.012 mol/L HCl (Vitrogen, Cohesion, Palo Alto, CA) and 10-fold concentrated DMEM without FBS and antibiotics. The pH of the solution was adjusted using 0.2 mol/L NaOH and 500 µL of this solution was added to 24-well plates. Twelve spheroids (six treated and six untreated in the control group) were implanted into the gel so that the spheroids were completely surrounded by the collagen matrix. After gelation at 37°C, the gel was overlaid with 500 µL supplemented DMEM and the inhibitor. When bacitracin was added for the first time, a concentration of 10 mmol/L of bacitracin was applied to saturate the collagen phase. During medium replacement, 5 mmol/L bacitracin in DMEM was used. Cell invasion distances were measured using an inverted phase-contrast light microscope with a micrometer in the ocular. Invasion was measured daily, and medium was renewed every 3rd day. To measure the invasion distance in the collagen gel, the same criteria as used in the migration assays were applied. Measurements were taken for 10 days. The longest treatment with bacitracin was 9 days. The experiment was done twice.
Statistical analyses. A migration distance of treated and untreated spheroid cells was compared with Student's t test (independent samples, two tailed). Type I error level was set to = 0.05.
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Results |
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PDI is expressed on the surface of glioblastoma cells. The surface localization of PDI on U373 glioblastoma cells was verified by flow cytometry. Compared with the controls, the flow cytometric analysis revealed a significant increase in fluorescence intensity in the nonpermeabilized U373 cells [mean fluorescence intensity (MFI), 86.37] compared with the control cells (MFI, 3.39; Fig. 5A ). The same cells were also studied by confocal microscopy, showing PDI to be expressed at the cell surface (Fig. 5A).
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PDI inhibition reduces tumor cell migration. Because PDI was expressed by the invasive tumor cells in vivo and by migrating glioma cells in vitro, we assessed the effect of a well-known PDI inhibitor, bacitracin (15, 16), on glioma cell migration and invasion.
Our previous experiments showed that PDI was stronger expressed in the low-generation, invasive tumors. We therefore tested whether the low-generation glioma spheroids were more sensitive to PDI inhibition by bacitracin than those derived from the high-generation tumors. We exposed both low- and high-generation spheroids to 5 mmol/L bacitracin, 4 hours after plating them into 24-well plates. The results show that cell migration from both low- and high-generation spheroids was inhibited by bacitracin and that the inhibition was stronger in cells derived from low-generation spheroids, as an almost complete inhibition of migration was obtained (Fig. 6A ). In contrast, the treated high-generation spheroids attached to the bottom and some cells migrated, but the migration distance was strongly reduced compared with the control group. The inhibitory effect of bacitracin on the high-generation spheroids was more pronounced after 3 days, and the difference in migration distance compared with the control group increased with time (Fig. 6A).
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To eliminate possible toxic effects of 5 mmol/L bacitracin exposure, we investigated whether its inhibitory effect on cell migration was reversible. We divided spheroids into four groups, six spheroids in each group. Control spheroids did not receive any treatment, whereas other groups were treated for 2, 4, and 7 days before bacitracin removal. The results show that removal of the inhibitor caused an immediate migration of glioma cells similar to that seen in the untreated group. Thus, the effect of 5 mmol/L bacitracin was reversible (Fig. 6C).
A concentration of 10 mmol/L bacitracin had a toxic effect on the spheroids, characterized by no attachment of spheroids to a plastic surface and spheroid dissociation (data not shown). Bacitracin (5 mmol/L) had a clear effect on both high-and low-generation spheroids, as well as on glioma cell line spheroids without causing toxicity (Fig. 6C). Bacitracin at the concentration of 2.5 mmol/L also reduced the glioma cell migration, but the effect was weaker than that seen by 5 mmol/L (Fig. 6D).
Because bacitracin is a chemical inhibitor that may unspecifically interfere with other intracellular processes not related to PDI, we also wanted to see if a PDI-specific biological inhibitor showed similar results to those obtained for bacitracin. We therefore tested the inhibitory effect of a monoclonal antibody against PDI on a highly invasive and tumorigenic glioblastoma cells U373 (11), which express high levels of PDI (data not shown). As shown in Fig. 6D, the monoclonal antibody inhibited cell migration in a dose-dependent manner. The inhibitory effect of the PDI antibody was significant (P 
0.05) but less than that observed using 5 mmol/L bacitracin.
Bacitracin and ß3 integrin antibody show additive effects on cell migration. The effects of ß1 and ß3 integrin blocking antibodies on glioma U373 cell migration were determined and the inhibitory effects were compared with those obtained using 2.5 mmol/L bacitracin. Blocking the ß1 integrin had no effect on cell migration (data not shown). In contrast, the blocking ß3 integrin antibody reduced migration similar to that observed using 2.5 mmol/L bacitracin. Moreover, when both bacitracin at the concentration of 2.5 mmol/L and ß3 integrin antibody at the dilution 1:100 were used together, the inhibitory effect was stronger than that observed if the agents were used separately (Fig. 6F).
Bacitracin inhibits glioma invasion in vitro. In addition to the inhibitory effect of bacitracin on glioma cell migration in vitro, we confirmed that bacitracin reduces glioma cell penetration into collagen gels, which indicates an inhibitory effect on glioma invasion in vitro. We applied bacitracin at a concentration of 5 mmol/L on six low-generation spheroids, placed in the collagen gel. There was clear inhibition of cell penetration into the collagen gel (Fig. 6E).
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Discussion |
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One of the proteins identified was PDI A6 precursor (synonyms: PDI P5, thioredoxin domain-containing protein 7, human P5). PDI A6 is a 440-amino acid protein, belonging to the PDI family. Human P5 contains two thioredoxin-like domains, like human PDI, and these domains share 47% amino acid sequence identity in PDI and P5. P5 belongs to the PDI protein family (24), and PDI is expressed on the cancer cell surface. We verified the presence of PDI on the cell surface using flow cytometry and confocal microscopy. We also verified that PDI was differentially expressed in the two phenotypes. It has been proposed that P5 is functionally related to PDI, which is a chaperone protein, involved in the protein quality control machinery (25). Its major role is in rearrangement of both intrachain and interchain disulfide bonds, where it acts catalytically to both initiate and reduce disulfide bonds. It has been postulated that PDI, on the cell surface, acts as a reductase cleaving the disulfide bonds of proteins attached to the cell surface and inside the cell it forms disulfide bonds (26). It may also control the function of certain extracellular proteins by regulating their red-ox state (27).
P5 cDNA was first isolated from a hydroxyurea-resistant hamster cell line (28). It has been proposed that P5 plays an important role in acquiring hydroxyurea resistance (29). It has also been shown that PDI is up-regulated in glial cells during hypoxia/brain ischemia, and a role of PDI in apoptosis resistance has been postulated (25, 30). Here, we identified this protein to be strongly expressed by the most invasive glioma phenotype.
Immunohistochemistry and immunoblots revealed that PDI was expressed in both phenotypes. However, there was a quantitative difference in its expression between the two phenotypes. In the low-generation tumors, PDI was extensively expressed throughout the tumor mass, whereas, in the high-generation tumors, PDI was confined to the tumor periphery and was not present in the angiogenic core. The same observations were made in the biopsy specimens obtained from patients, where PDI was expressed in the tumor periphery. PDI was also expressed by migrating tumor cells in vitro, suggesting that PDI has a functional role in glioma cell migration. To address this issue, we exposed the cells derived from the two phenotypes to a PDI-specific inhibitor, bacitracin (15). Interestingly, bacitracin had an inhibitory effect on migration and invasion of tumor cells derived both from the low- and high-generation tumors. However, the effect was strongest on the invasive phenotype (Fig. 6A and B).
It has been suggested that PDI functions at the cell surface, where it may be involved in disulfide exchange required for cell-mediated adhesion by integrins (31). Several integrins have been linked to glioma cell invasion, and a particular focus has been on the ß1 and ß3 integrins (1). Interestingly, it has been shown that PDI can mediate conformational changes in both ß1 and ß3 integrins, which may lead to cell adhesion to a particular substrate (32). Here, we show that a blocking ß3 integrin antibody has an inhibitory effect on U373 cell migration in vitro and that this effect does not abrogate further migration inhibition by bacitracin.
In addition, a direct intervention of PDI in cellular adhesion has been shown in retina cells from chicken embryo, where a cell surface adhesion protein was identified to be PDI (16). In our experiments, we have shown that incubation of glioblastoma cells with PDI antibody inhibits adhesion of U373 cells to the plastic surface, indicating a role of PDI in glioma cell adhesion.
The fact that a PDI-specific antibody also inhibited cell migration suggests a functional role of PDI in glioma cell migration and invasion. The inhibitory effect using the PDI antibody was less than that observed using bacitracin. This observation is consistent with earlier reports showing a 49% inhibition of PDI activity by using the PDI antibody (15).
In conclusion, using a novel human glioblastoma xenograft model and proteomics screening technology, we were able to identify a novel protein, PDI, which is strongly expressed by invasive glioma cells. Moreover, inhibition of PDI led to reduced glioma cell migration and invasion. PDI may therefore represent a potential therapeutic target in malignant brain tumors.
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Acknowledgments |
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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.
We thank Erna Finsås for technical assistance with protein electrophoresis and Western blot, Tove Johansen for the help with cell culture, Tore Jacob Raa and Linda Vabø for taking care of the animals, and colleagues at PROBE for all the help and expertise in doing MS.4
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Footnotes |
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Received 12/27/05. Revised 6/21/06. Accepted 8/ 4/06.
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2ß1. Blood 2003;102:2085–92.This article has been cited by other articles:
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  P. E. Lovat, M. Corazzari, J. L. Armstrong, S. Martin, V. Pagliarini, D. Hill, A. M. Brown, M. Piacentini, M. A. Birch-Machin, and C. P.F. Redfern Increasing Melanoma Cell Death Using Inhibitors of Protein Disulfide Isomerases to Abrogate Survival Responses to Endoplasmic Reticulum Stress Cancer Res., July 1, 2008; 68(13): 5363 - 5369. [Abstract] [Full Text] [PDF]
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D. M. Townsend, L. He, S. Hutchens, T. E. Garrett, C. J. Pazoles, and K. D. Tew NOV-002, a Glutathione Disulfide Mimetic, as a Modulator of Cellular Redox Balance Cancer Res., April 15, 2008; 68(8): 2870 - 2877. [Abstract] [Full Text] [PDF]
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 P. C. Huszthy, D. Goplen, F. Thorsen, H. Immervoll, J. Wang, A. Gutermann, H. Miletic, and R. Bjerkvig Oncolytic Herpes Simplex Virus Type-1 Therapy in a Highly Infiltrative Animal Model of Human Glioblastoma Clin. Cancer Res., March 1, 2008; 14(5): 1571 - 1580. [Abstract] [Full Text] [PDF]
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 Y. Wang, Y. Ma, B. Lu, E. Xu, Q. Huang, and M. Lai Differential Expression of Mimecan and Thioredoxin Domain Containing Protein 5 in Colorectal Adenoma and Cancer: A Proteomic Study Experimental Biology and Medicine, October 1, 2007; 232(9): 1152 - 1159. [Abstract] [Full Text] [PDF]
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