Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases

Abstract

Alterations of the epidermal growth factor receptor (EGFR) gene occur frequently in human malignant gliomas. The most common of these is deletion of exons 2-7, resulting in truncation of the extracellular domain (DeltaEGFR or EGFRvIII), which occurs in a large fraction of de novo malignant gliomas (but not in progressive tumors or those lacking p53 function) and enhances tumorigenicity, in part by decreasing apoptosis through up-regulation of Bcl-XL. Here, we demonstrate that the DeltaEGFR concomitantly confers resistance to the chemotherapeutic drug cisplatin (CDDP) by suppression of CDDP-induced apoptosis. Expression of Bcl-XL was elevated in U87MG.DeltaEGFR cells prior to and during CDDP treatment, whereas it decreased considerably in CDDP-treated parental cells. CDDP-induced activation of caspase-3-like proteases was suppressed significantly in U87MG.DeltaEGFR cells. These responses were highly specific to constitutively kinase-active DeltaEGFR, because overexpression of kinase-deficient DeltaEGFR (DK) or wild-type EGFR had no such effects. Correspondingly, DeltaEGFR specific tyrosine kinase inhibitors reduced Bcl-XL expression and potentiated CDDP-induced apoptosis in U87MG.DeltaEGFR cells. Ectopic overexpression of Bcl-XL in parental U87MG cells also resulted in suppression of both caspase activation and apoptosis induced by CDDP. These results may have important clinical implications for the use of CDDP in the treatment of those malignant gliomas expressing DeltaEGFR.

Figure 1

(A) Quantitation of EGFR species in cells. Western blot analysis of expression (Lower) and autophosphorylation (Upper) of wt EGFR (♦) and ΔEGFR (•) in U87MG, U87MG.ΔEGFR, U87MG.DK, and U87MG.wtEGFR cells grown in medium containing 10% serum. Low and similar levels of endogenous wt EGFR expression also was detected in U87MG, U87MG.ΔEGFR, and U87MG.DK at longer exposures (data not shown). (B) Survival of U87MG (□), U87MG.ΔEGFR (⋄), U87MG.DK (○), and U87MG.wtEGFR (▵) cells in response to varying amounts of cisplatin (CDDP). Cells were plated in triplicate 60-mm dishes and treated with medium containing various concentrations of CDDP for 1 hr, followed by incubation with fresh medium for 10–12 days. Numbers of colonies were counted after Giemsa staining. Results were reproduced in four independent experiments. [Bars = SD (some bars are too small to be visualized).]

Figure 2

Cells expressing ΔEGFR are diminished in their apoptotic response to CDDP. Cells were seeded on coverslips and treated with 5 μg/ml of CDDP for 2 days, and then TUNEL assays were performed. The percentage of TUNEL-positive cells induced by CDDP treatment was assessed by counting more than 400 cells per coverslip. □, U87MG; ▪, U87MG.ΔEGFR; ▨, U87MG.DK; ▧, U87MG.wtEGFR. Values represent means from three independent experiments, each performed in triplicate. ∗∗∗, Significantly different (P < 0.001) from the value for the U87MG.ΔEGFR cells treated with CDDP. (Bars = SE.)

Figure 3

Cells expressing ΔEGFR have higher initial and sustained levels of Bcl-X_L. Western blot analysis of Bcl-X_L expression in U87MG, U87MG.ΔEGFR, U87MG.DK, and U87MG.wtEGFR cells was performed after CDDP treatment. Total cell lysates were prepared at the various time points indicated. For each sample, 20 μg of lysate protein was used. Results were reproduced in three independent experiments.

Figure 4

ΔEGFR expression causes reduced activation of caspase-3-like proteases in response to CDDP treatment. (A) Total cell lysates were prepared 2 days after treatment with 5 μg/ml of CDDP. Samples were assayed for protease activity by using the peptide substrate Ac-DEVD-pNA. For inhibition of the protease activity, 10 μM Ac-DEVD-CHO was added to the reaction mixture before the addition of substrate. Cells also were treated with the plasma membrane-soluble caspase inhibitor Z-Asp-CH₂-DCB (200 μM) before and during CDDP treatment. □, U87MG; ▪, U87MG.ΔEGFR; ▨, U87MG.DK; ▧, U87MG.wtEGFR. Values represent the means of seven independent experiments (two for the Z-Asp-CH₂-DCB experiment). ∗∗∗, Significantly different (P < 0.001) from the value for U87MG.ΔEGFR cells treated with CDDP. (Bars = SE.) (B) Proteolytic cleavage of PARP after CDDP treatment. For each sample, 20 μg total clarified protein lysate was loaded onto SDS/PAGE gels, electrophoresed, transferred to membranes, and probed with anti-PARP mAbs. FL, full-length; CF, cleaved fragment.

Figure 5

Overexpression of Bcl-X_L inhibits the caspase-3-like protease activation and induction of apoptosis caused by CDDP treatment. (A) Western blot analysis showing expression levels of Bcl-X_L in U87MG Bcl-X_L-overexpressing clones (Bcl-X_L-6, -9, -13, -11, -12, and -8) and empty vector-transfected clones (SFFV-2 and -5). U87MG and U87MG.ΔEGFR cells were treated with 5 μg/ml of CDDP for 2 days. For each sample, 20 μg total cell lysate was used. (B) Caspase-3-like protease activity of the Bcl-X_L-overexpressing clones 2 days after treatment with 5 μg/ml CDDP measured as described in Fig. 4A. Values are means of two to six independent experiments. (Bars = SE.) (C) Apoptosis rate of Bcl-X_L-overexpressing clones 2 days after treatment with 5 μg/ml of CDDP determined by TUNEL assay. Values represent means from two to three independent experiments in triplicate. (Bars = SE.)

Figure 6

Modulation of CDDP-induced apoptosis in U87MG.ΔEGFR cells by various tyrphostin-type tyrosine kinase inhibitors. (A) Reduced Bcl-X_L expression upon exposure to tyrphostin AG1478. U87MG.ΔEGFR cells were treated with the ΔEGFR-selective tyrphostin AG1478 (15 μM) with or without CDDP (5 μg/ml), and total cell lysates were prepared. For each sample, 20 μg lysate was used. (B) Increased apoptosis induced by combination treatment by using ΔEGFR-selective tyrphostins (AG1478 or AG1517) and CDDP. U87MG.ΔEGFR cells were treated with or without 5 μg/ml CDDP for 2 days in the presence of the ΔEGFR-selective tyrphostins (AG1478 or AG1517) or the nonspecific, less potent tyrphostins (AG1479 or AG1536) at the concentrations indicated. For control, the vehicle (dimethyl sulfoxide) was used. Apoptosis rate was determined by TUNEL assays from triplicate coverslips. Experiments were repeated independently two to three times, with similar results.