Biochemical correlates of mTOR inhibition by the rapamycin ester CCI-779 and tumor growth inhibition

Lorina Dudkin, Michael B. Dilling, Pamela J. Cheshire, Franklin C. Harwood, Melinda Hollingshead, Susan G. Arbuck, Robert Travis, Edward A. Sausville, Peter J. Houghton

Research output: Contribution to journalArticlepeer-review

244 Scopus citations


The rapamycin ester, CCI-779, potently inhibits cell growth in vitro, inhibits tumor growth in vivo, and is currently in Phase I clinical trials. To further understand the relationship between plasma systemic exposure and inhibition of the target Ser/Thr kinase, mTOR/FRAP, two assays have been developed. The first assay involves determination of the 4E suppressor protein (4E-BP1) bound to eukaryotic initiation factor 4E (eIF4E), and the second is direct Western analysis of phosphorylation of residue Thr70 of 4E-BP1. Under normal growth conditions in vitro, rapamycin caused rapid association of 4E-BP1 with eIF4E within 1 h in Rh30 and GC3 human tumor cells. Association was persistent up to 16 h. In mice, administration of rapamycin (5 or 20 mg/kg) caused rapid association of 4E-BP1 with eIF4E within 4 h in both human colon adenocarcinoma GC3, and rhabdomyosarcoma Rh30 xenografts. Using phospho-specific antibody against Thr70 of 4E-BP1, rapid and persistent dephosphorylation within 30 min of exposure to rapamycin was detected in Rh18 rhabdomyosarcoma cells. Evaluation of CCI-779 against Rh18 xenografts showed this tumor to be growth inhibited at daily dose levels of ≥8.7 mg/kg. Because immunoblotting may be more suitable for assaying tumor biopsy tissue, a "blinded" comparison between the effect of CCI-779 on Thr70 phosphorylation and growth inhibition of human tumor xenografts was undertaken. Mice were treated daily for 5 days with CCI-779 (20 mg/kg/day) or with drug vehicle, and tumor diameters were measured. Tumors were excised 1 h after the final administration and frozen, and phospho Thr70 was determined by Western blot analysis. The correlation coefficient for decreases in Thr70 phosphorylation and growth inhibition was high (r2, 0.99). The results indicate that an assay of decreases in phosphorylation of Thr70 of 4E-BP1 may be a useful surrogate for determining the inhibition of mTOR activity in tumor specimens.

Original languageEnglish (US)
Pages (from-to)1758-1764
Number of pages7
JournalClinical Cancer Research
Issue number6
StatePublished - 2001
Externally publishedYes

ASJC Scopus subject areas

  • Oncology
  • Cancer Research


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