Abstract
OBJECTIVE To investigate the correlation between the sensitivity to the tumor necrosis factor-related apoptosis inducing ligand(TRAIL) and the level of expression of the death receptor 5(DR5)on the surface of tumor cells.
METHODS Anti-DR5 mAbs were used to directly detect the level of expression of DR5 on the surface of tumor cells. Using a TRAIL apoptosis kit and flow cytometry, the sensitivity of the tumor cells to TRAIL-induced apoptosis was determined and the correlation between DR5 expression and sensitivity to TRAlL analyzed.
RESULTS The expression level of DR5 on the surface of different tumor cells was as follows: 97.9% in U937 cells, 95.1% in Jurkat cells, 93.8% in SW480 cells, 86.2% in HCT116 cells, 64.2% in HL-60 cells, 46.6% in Hela cells and 13.1 % in K562 cells. The TRAIL-induced apoptotlc rate was 72.6% in U937 cells, 85.2% in Jurkat cells, 78.6% in SW480 cells, 70.2% in HCT116 cells, 60.1% in HL-60 cells, 45.4% in Hela cells and 12.3% in K562 cells. Statistical analysis showed there was a significant positive correlation (r=0.997, P < 0.001) between DR5 expression and sensitivity to TRAIL.
CONCLUSION The sensitivity of tumor cells to TRAIL is related to the level of expression of DR5 on the surface of tumor cells. These results confirm the importance of DR5 expression for induction of apoptosis by TRAIL.
keywords
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)[1] can induce apoptosis in most tumors, but has no effect on most normal cells.[2] Consequently this ligand is of great interest to cancer researchers. The means by which TRAIL induces apoptosis of tumor cells is by first binding to one of its receptors. At present, five TRAIL receptors have been identified, among which TRAIL-R1 (death receptor 4, DR4) and TRAIL-R2 (death receptor 5, DR5) can convey a death signal and initiate apoptosis of tumor cells.[3] The other three receptors, i.e. (1) a soluble receptor (OPG), (2) TRAIL-R3 (decoy receptor 1, DcRl with no intracellular death structural domain) and (3) TRAIL-R4 (decoy receptor 2, DcR2; DD no integral) cannot transfer a death signal for TRAIL.[4] Under normal physiological conditions, the affinity of DR5 with TRAIL is significantly stronger than with DR4,[5] so the role of DR5 is especially important in both conveying the death signal of TRAIL and inducing tumor-cell apoptosis. In order to study the correlation between TRAIL inducd-apoptosis of tumor cells and the level of expression of DR5 on the surface of tumor cells, anti-DR5 monoclonal antibodies were developed to evaluate the expression of DR5 on various tumor cell lines. The sensitivity of TRAIL to induce apoptosis was determined by using flow cytometry.
Materials and Methods
Anti-DR5 monoclonal antibodies
Prepared in our laboratory.[6]
The cell lines
Jurkat and U937 cells were cultivated in RPMI 1640 culture mediumcontaining 2 mmol/L L-glutamine, 1.5 g/L sodium bicarbonate, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 10% FBS, 100 U/ml penicilin and 100 μg/ml streptomycin. SW480 cells were cultivated in Leibovitz’s L-15 medium, with the addition of 2 mmol/L L-glutamine and 10% FBS. HCT 116 cells were grown in Mc-Coy’s 5a medium, with the addition of 1.5 mmol/L L-glutamine and 10% FBS. K562 and HL-60 cells were grown in Iscove’s DMEM culture medium, adding 4 mmol/L glutamine, 1.5 g/L sodium bicarbonate and 20% FBS. Hela cells were cultivated in DMEM culture medium containing 10% FBS, 2 mmol/L L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.
Determination of the expression level of DR5 on the surface of tumor cells
The various cells to be tested were washed three times with PBS, and 1 × 105/cells added to each well of a 96-well culture plate. Anti-DR5 mAbs (1 μg) were placed in each well and the plate kept on ice for 30 min, Precooled PBS was used to wash the cells three times; 1 μg of FTTC labelled goat anti-mouse IgG (Jackson Immuno Research) was added and the plate maintained on ice for 30 min. After three washings with PBS the cells were analyzed by flow cytometry (FACScan, Becton Dickinson).
Determination of the apoptosis rate
A TRAIL apoptosis kit (Upstate Biotech) was used. Fifty μl of medium was added to each well of a U-type 96-well plate followed by 50 μl of TRAIL (with a final concentration of 100 ng/ml). A 50 μaliquot was withdrawn from the No.l well and transferred to the No.2 well followed by similar serial dilutions throughout the plate.To each well 50 μl of potentiator (mouse anti-TRAIL monoclonal antibody IgGl) was addedto give a final concentration of 1.5 μg/ml followed by the addition of 100 μl of cell suspension to be tested (1 × 106/ml of final concentration). The cells were cultured for 12 h and washed three times with 100 μl of PBS after which10 μl of PI was added to each tube. Flow cytometry was used to detect the apoptotic rate.
Results
The level of expression of DR5 on the surface of tumor cells
Using anti-DR5 mAbs and flow cytometry the expression level of DR5 was determined directly on the surface of various tumor cells. The expression rate of DR5 was as follows: 97.9% in U937 cells, 95.1% in Jurkat cells, 93.8% in SW480 cells, 86.2% in HCT 116cells, 64.2% in HL-60 cells, 46.6% in Hela cells and 13.1% in K562 cells. There was big difference in the expression level of DR5 on surface of various tumor cells, e.g., the U937, Jurkat, SW480 and HCT 116 cells showed a high level of expression, HL-60 andHela cells showed an intermediate level, while the K562 cells only showed a low levelofexpression of DR5(Figs.1,2).
Expression of DR5 on the surface of Jurkat cells
Expression of DR5 on the surface of various tumor cells and TRAIL inducing apoptosis.
The rate of apoptosis induction by TRAIL
The apoptotic status of seven different tumor cell lines were determined using a TRAIL apoptosis kit. The apoptosis rate for the various cell lines was as follows: 85.2% in Jurkat cells, 78.6% in SW480 cells, 72.6% in U937 cells, 70.2% in HCT 116 cells, 60.1% in HL-60 cells, 45.4% in Hela cells and 12.3% in K562 cells (Figs.2,3). The sensitivity of different tumor cell lines to TRAIL varied greatly. The order of sensitivity to the effect of TRAIL from greatest to least was as follows: Jurkat, SW480, U937, HCT 116, HL-60 and Hela cells.
TRAIL-induced apoptosis of Jurkat cells
The relationship between the expression level of DR5 on the surface of tumor cells and the Induction of apoptosis by TRAIL
To investigate whether the sensitivity of tumor cells to TRAIL was related to the level of DR5 expression, a statistical analysis was conducted to determine the level ofcorrelation, with the result showing a positive correlation (r = 0.997, P < 0.001; Fig.4).
Analysis of the relationship between the level of DR5 expression on the surface of tumor cells and TRAlL-induced apoptosis.
Discussion
TRAIL has no effect on the majority of normal cells, though it can induce apoptosisof tumor cells. This special selectivity allows it to be of potential use for tumor therapy. However the question remains as to why there is such a big difference for TRAILinduced apoptosis between normal cells and various cell lines. This difference cannot be only explained by the expression of the induced receptor, FLIP (PLICE-inhibitory protein), etc.[7] Of the five different receptors of TRAIL, only DR4 and DR5can mediate the apoptotic signal for TRAIL, resulting in apoptosis. No matter how the apoptotic signal is conveyed, TRAIL must first combine with its receptor before inducing apoptosis. Therefore, the level of expression and function of the TRAIL receptors on the cell surface is of utmost importance to TRAIL-mediated apoptosis. Many studies have been conducted to examine the relation between the apoptotic action of TRAIL and its receptors. Because of a lack of effective monoclonal antibodies for the anti-TRAIL death receptor, RT-PCR determinations for mRNA or receptor protein have been principally used to study the TRAIL-death receptor, but these methods cannot thoroughly measurethe level of expression and function of the cell surface receptor. Kim, et al. [8] reported, using the above methods, that the tumor cell sensitivity to TRAIL was principally related to the level of expression of DR4 but not related to DR5 expression, which is not consistant with the fact that, under physiological conditions at 37°C,[5] there was a higher affinity of TRAIL for DR5, one of the fiveTRAIL receptors.
To study the level of expression and function of DR5, as wellas the relationship between its expressed amount and TRAIL-inducing apoptosis, monoclonal antibodies which can combine with specificity for DR5 were developed. High-levels of DR5 expression were detected in cell lines sensitive to TRAIL, such as Jurkat, U937,SW480 and HCT 116, etc. At the same time there was a positive correlation between theexpression level of tumor cell surface DR5 and the sensitivity to TRAIL-inducing apoptosis. These findings are of importance to the understanding of the anti-cancer action of TRAIL and for guidance in the clinical application of TRAIL. Kim, et al.[8] came to a different conclusion because they had found a high sensitivity of SW480 and HCT 116 cells to TRAIL but did not report that there was a high-level of expression of DR5. Mitsiades, et al.[7] measured the amount of protein expression ofDR4 and DR5 in ten Ewing’s sarcoma cells and determined their sensitivity to TRAIL, using Western blotting. Their results indicated that nine cell lines which were sensitive to TRAIL showed protein expression of DR4 and DR5, and the remaining one which was resistant to TRAIL had expression of only DR4. After the DR5 gene was transfected into the resistant TRAIL cells, they expressed DR5 and sensitivity to TRAIL. Four cell lines sensitive to TRAIL were identified by using monoclonal antibodies and flow cytometry, with the results that there was expression of DR5 on the surface of all the cells, while the expression of DR4 occurred in only two cell lines. These results are in accordance with our conclusions. Moreover, we also demonstrated that TRAIL-mediated apoptosis of Jurkat cells can be blocked by anti-DR5 mAbs, [9] which can further explain the correlation between the sensitivity of tumor cells to TRAIL and the expression of DR5 on surface of tumor cells. The results of this study show that DR5 plays a most important role for induction of apoptosis by TRAIL.
Footnotes
This work was supported by Henan Provincial Foundation for Financial Aids to the Prominent Talents (No. 0321001800); Henan Provincial Foundation for Aids to Technology Innovation and Talents Project of Medical Science(No. 2002-119).
- Received October 30, 2004.
- Accepted March 23, 2005.
- Copyright © 2005 by Tianjin Medical University Cancer Institute & Hospital and Springer
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