Increased Expression of Mcl-1 Is Required for Protection against Serum Starvation in Phosphatase and Tensin Homologue on Chromosome 10 Null Mouse Embryonic Fibroblasts, but Repression of Bim Is Favored in Human Glioblastomas

Mark Austin,Simon J Cook

doi:10.1074/jbc.m508187200

Abstract

Inactivating mutations in the tumor suppressor gene phosphatase and tensin homologue on chromosome 10 (PTEN) result in elevated levels of phosphatidylinositol (3,4,5)-trisphosphate, activation of protein kinase B (PKB), and protection against apoptotic insults such as withdrawal of survival factors. Protection may arise through the inhibition of the pro-apoptotic protein Bim, which is normally repressed by a PKB-dependent mechanism. Here we show that PTEN-/- immortalized mouse embryonic fibroblasts (MEFs) exhibit elevated PKB phosphorylation and are resistant to serum withdrawal-induced death, but exhibit normal Bim expression following withdrawal of serum. In contrast, expression of Mcl-1, a prosurvival member of the Bcl-2 family, was elevated in PTEN-/- MEFs. Transient or stable overexpression of Mcl-1 in PTEN+/- MEFs conferred resistance to serum withdrawal, whereas ablating expression of Mcl-1 in PTEN-/- MEFs, using RNA interference, abolished their resistance to serum withdrawal-induced apoptosis. To determine if Mcl-1 is selected for overexpression in human tumors we examined human glioblastoma cell lines but found that loss of PTEN had no effect on Mcl-1 expression. In contrast, two of three PTEN-/- glioblastoma cell lines exhibited low expression of Bim, which was refractory to serum withdrawal. These results indicate that the resistance of PTEN-/- MEFs to serum withdrawal is largely due to the up-regulation of Mcl-1 but that loss of PTEN in tumor cell lines is more complex and may favor de-regulation of different apoptotic regulators such as Bim.

Highlights

phosphatase and tensin homologue on chromosome 10 (PTEN) locus (10q23) is associated with many high grade glioblastomas and endometrial tumors as well as some melanoma, prostate, and breast carcinomas [1]
protein kinase B (PKB) Phosphorylation Is Elevated in Immortalized PTENϪ/Ϫ Mouse Embryonic Fibroblasts—We sought to characterize the effect of serum withdrawal on immortalized mouse embryonic fibroblasts (MEFs) cell lines derived from mice heterozygous or homozygous null for PTEN [13]
To simplify interpretation we examined serum withdrawal-induced apoptosis, because (a) this is a relatively benign form of cell stress that doesn’t involve DNA damage, (b) serum growth factors would be expected to determine in part the activation state of the PKB pathway, and (c) loss of growth factor dependence for survival is a hallmark of cancer cells [36]

Summary

EXPERIMENTAL PROCEDURES

Materials—Cell culture reagents were purchased from Invitrogen. The following antibodies were used throughout this study: PTEN (6H2.1) was from Cascade Bioscience; ERK1/2 and Bcl-XL were from BD Biosciences Pharmingen; Bim was from Chemicon; Bmf was from Calbiochem; Actin, ERK2, Mcl-1, Bcl-2, Bak, and Bax (N-20) were from Santa Cruz Biotechnology; phospho-ERK1/2 (Thr-202/Tyr-204), cleaved caspase-3 PKB, phospho-PKB (Thr-308), and phospho-PKB (Ser-473) were from Cell Signaling Technologies. PTENϩ/Ϫ MEFs overexpressing HA-Mcl-1 were derived by transfection (FuGENE 6, Roche Applied Science), Zeocin selection (300 ␮g/ml), and ring cloning after limiting dilution. PTENϪ/Ϫ MEFs expressing shRNA against Mcl-1 were derived by transfection (FuGENE 6, Roche Applied Science), Hygromycin B selection (500 ␮g/ml) and ring cloning after limiting dilution. PTENϩ/Ϫ cells were transfected with 10 ␮g of pcDNA:HA:Mcl-1 or empty vector and 2 ␮g of pcDNA:EGFP-spectrin (Paul Coffer, University of Utrecht), using a calcium phosphate precipitation protocol to ensure that EGFP-expressing cells were expressing HA:Mcl-1. Cells were stained with propidium iodide and the EGFP-spectrin-expressing population was analyzed for sub-G1 DNA content by flow cytometry. (Q)RT-PCR was performed according to the protocol supplied with the TaqMan® reverse transcription reagents (Applied Biosystems) as described previously [18]. For mouse Mcl-1 we used 5Ј-TGTAAGGACGAAACGGGACT-3Ј as the forward primer and 5Ј-AAAGCCAGCAGCACATTTCT-3Ј as the reverse primer. For human ␤-Actin we used 5Ј-TGTTTGAGACCTTCAACACC-3Ј as the forward primer and 5Ј-TAGGAGCCAGAGCAGTAATC-3Ј as the reverse primer

RESULTS

Deletion of PTEN in Human Glioblastoma Cells Does Not Affect

DISCUSSION

ADDITIONS AND CORRECTIONS

Additions and Corrections