STUDIES ON THE MECHANISM OF RESISTANCE TO RAPAMYCIN IN HUMAN CANCER-CELLS

Rapamycin is a potent cytostatic agent that arrests cells in the G1 ph ase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rap amycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70(S6k)) and dephosphorylation of pH acid stable prote in 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 [C-14 ]rapamycin were similar in sensitive and resistant cells, with all cel ls examined demonstrating a stable tight binding component. Western an alysis showed levels of mTOR were similar in each cell line (<2-fold v ariation). The activity of p70S6k, activated downstream of mTOR, was s imilar in four cell lines (range, 11.75-41.8 pmol/2 x 10(6) cells/30 m in), but activity was equally inhibited in cells that were highly resi stant to rapamycin-induced growth arrest. Rapamycin equally inhibited serum-induced phosphorylation of pH acid stable protein I in Rh1 (intr insically resistant) and sensitive Rh30 cells. In serum-fasted Rh30 an d Rh1 cells, the addition of serum rapidly induced c-MYC (protein) lev els. Rapamycin blocked induction in Rh30 cells but not in Rh1 cells. S erum-fasted Rh30/rapa10K cells, selected for high level acquired resis tance to rapamycin, showed greater than or equal to 10-fold increased c-MYC compared with Rh30. These results suggest that the ability of ra pamycin to inhibit c-MYC induction correlates with intrinsic sensitivi ty, whereas failure of rapamycin to inhibit induction or overexpressio n of c-MYC correlates with intrinsic and acquired resistance, respecti vely.