4E-binding Proteins, the Suppressors of Eukaryotic Initiation Factor 4E, Are Down-regulated in Cells with Acquired or Intrinsic Resistance to Rapamycin

Michael B Dilling,Glen S Germain,Lorina Dudkin,Arun L Jayaraman,Xiongwen Zhang,Franklin C Harwood,Peter J Houghton

doi:10.1074/jbc.m110782200

Michael B Dilling, Glen S Germain + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110782200

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Abstract

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.

Highlights

Rapamycin is a macrocyclic lactone antibiotic that potently inhibits the activation of T cells and the growth of many malignant cells in culture [1,2,3,4,5]
These results suggest that the 4E-binding protein (4E-BP):eIF4E ratio is an important determinant of rapamycin resistance and controls certain aspects of the malignant phenotype
The Rate of 4E-BP Synthesis Is Reduced in Resistant Cells—We investigated whether steady-state levels of 4E-BP were reduced in resistant clones because of decreased synthesis or because of increased degradation

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—The rhabdomyosarcoma cell lines Rh30 and Rh18 [33, 37] and the G2 glioblastoma cell line [37] were established from pediatric tumors. Samples were incubated (37 °C, 5% CO2) for up to 4 h, washed twice with 10 ml of cold PBS, and lysed at 4 °C for 1 h in 1 ml of cell lysis buffer (Cell Signaling, Beverly, MA) containing protease inhibitors. Western Analysis of 4E-BP1—Cultured cells at a logarithmic phase of growth were briefly washed with cold PBS, and samples containing 1.5 ϫ 106 cells were lysed on ice in 75 ␮l of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na3VO4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 tablet of protease inhibitor mixture (Roche CompleteTM Mini). Colonies were enumerated by using an AlphaImager 2000TM (Alpha Innotech Corp.)

RESULTS

Colony count

DISCUSSION