Studies on the Mechanism of Resistance to Rapamycin in Human Cancer Cells
- Hajime Hosoi1,1,
- Michael B. Dilling1,
- Linda N. Liu1,
- Mary K. Danks1,
- Takuma Shikata1,
- Aleksander Sekulic2,
- Robert T. Abraham2,
- John C. Lawrence, Jr.3 and
- Peter J. Houghton1
- 1Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 (H.H., M.B.D., L.N.L., M.K.D., T.S., P.J.H.), 2Department of Immunology, Mayo Foundation, Rochester, Minnesota 55905 (A.S., R.T.A.), and 3Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908 (J.C.L.)
Abstract
Rapamycin is a potent cytostatic agent that arrests cells in the G1 phase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rapamycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70S6k) and dephosphorylation of pH acid stable protein I (eukaryotic initiation factor 4E binding protein) were examined. We show that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold). Accumulation and retention of [14C]rapamycin were similar in sensitive and resistant cells, with all cells examined demonstrating a stable tight binding component. Western analysis showed levels of mTOR were similar in each cell line (<2-fold variation). The activity of p70S6k, activated downstream of mTOR, was similar in four cell lines (range, 11.75–41.8 pmol/2 × 106cells/30 min), but activity was equally inhibited in cells that were highly resistant to rapamycin-induced growth arrest. Rapamycin equally inhibited serum-induced phosphorylation of pH acid stable protein I in Rh1 (intrinsically resistant) and sensitive Rh30 cells. In serum-fasted Rh30 and Rh1 cells, the addition of serum rapidly induced c-MYC (protein) levels. Rapamycin blocked induction in Rh30 cells but not in Rh1 cells. Serum-fasted Rh30/rapa10K cells, selected for high level acquired resistance to rapamycin, showed ≥10-fold increased c-MYC compared with Rh30. These results suggest that the ability of rapamycin to inhibit c-MYC induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-MYC correlates with intrinsic and acquired resistance, respectively.
Footnotes
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Send reprint requests to: Peter J. Houghton, Ph.D., Dept. of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. E-mail:peter.houghton{at}stjude.org
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↵1 Current affiliation: Kyoto Prefectural University of Medicine, Kyoto 605, Japan.
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This work is supported in part by American Cancer Society Grants RPG-95–031-03-DHP (P.J.H.) and RPG-95–040-03 (R.T.A.) and United States Public Health Service Awards CA23099, 5T32CA09346 (L.N.L.), CA21675 (Cancer Center CORE), by a grant from Wyeth-Ayerst, and by American Lebanese, Syrian Associated Charities (ALSAC).
- Abbreviations:
- FKBP
- FK-binding proteins
- IL
- interleukin
- RMS
- rhabdomyosarcoma
- p70S6k
- ribosomal p70 S6 kinase
- IGF
- insulin-like growth factor
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- NF-AT
- nuclear factor of activated T cells
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
- TBST
- Tris-buffered saline/Tween 20
- PMSF
- phenylmethylsulfonyl fluoride
- PBS
- phosphate-buffered saline
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- SSC
- standard saline citrate
- PHAS-I
- pH acid stable protein I
- 4E-BP1
- eukaryotic initiation factor 4E binding protein
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- Received March 16, 1998.
- Accepted August 10, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
