ABSTRACT
Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21(Cip1)arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21(Cip1) independent of cell cycle arrest. Rapamycin induces p21(Cip1) binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21(Cip1).
Subject(s)
Apoptosis/drug effects , Cyclins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Sirolimus/pharmacology , Tumor Suppressor Protein p53/metabolism , Adaptor Proteins, Signal Transducing , Adenoviridae/genetics , Carrier Proteins/metabolism , Cell Cycle/drug effects , Cell Cycle Proteins , Cell Line , Culture Media, Serum-Free , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Dose-Response Relationship, Drug , Drug Resistance , Enzyme Activation , Fibroblasts , G1 Phase , Gene Expression Regulation , Humans , MAP Kinase Kinase Kinase 5 , MAP Kinase Kinase Kinases/metabolism , Models, Biological , Mutation , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Rhabdomyosarcoma/pathology , Time Factors , Tumor Suppressor Protein p53/geneticsABSTRACT
To determine whether inhibition of either the ribosomal p70 S6 kinase or eukaryotic initiation factor (eIF) 4E pathways downstream of the mammalian target of rapamycin, mTOR, contributes to rapamycin-induced growth arrest, clones of Rh30 rhabdomyosarcoma cells were selected for rapamycin resistance. Expression of c-Myc and anchorage-independent growth were enhanced in resistant cells. Resistance was unstable in each of three clones characterized. In resistant cells, as compared with parental cells, approximately 10-fold less 4E-binding protein (4E-BP) was bound to eIF4E, and total cellular 4E-BP was markedly reduced. Levels of eIF4E were unchanged. Steady-state levels of 4E-BP transcript remained unaltered, but the rate of 4E-BP synthesis was reduced in resistant cells. In cells that reverted to rapamycin sensitivity, levels of total 4E-BP returned to those of parental cells. Compared with parental cells, resistant clones had either similar or lower levels and activity of ribosomal p70 S6 kinase, but c-Myc levels were elevated in both resistant and revertant clones. Several colon carcinoma cell lines with intrinsic rapamycin resistance were found to have low 4E-BP:eIF4E ratios. In stable clones of HCT8 carcinoma engineered to overexpress 4E-BP, rapamycin sensitivity increased markedly (>1000-fold) as 4E-BP expression increased. These results suggest that the 4E-BP:eIF4E ratio is an important determinant of rapamycin resistance and controls certain aspects of the malignant phenotype.
