The disruption of the cell wall biosynthetic machinery is thought to

human lung cancer H460 cells were treated with increasing concentrations of rapamycin for 45 min. Phosphorylation of Bad was analyzed by Western blot using phospho specific Bad antibodies. reveal that inhibition of mTOR by rapamycin Bortezomib in increased Bad phosphorylation at S112 and S136 but not S155 in association with activation of ERK1/2 and Akt. As expected, decreased phosphorylation of mTOR, p70S6K or 4EBP1 was observed following rapamycin treatment, indicating that mTOR kinase activity was inhibited. It has already been established that phosphorylation of Bad at S112 or S136 inactivates its proapoptotic function. Our findings thus suggest that rapamycin induced Bad phosphorylation may lead to the loss of death promoting activity of Bad and thereby contribute to the resistance of human lung cancer cells to rapamycin. Similar were also obtained in H157 cells. This confirms Cellular differentiation that rapamycin induced Bad phosphorylation is not limited to a specific cell type. Rapamycin resistance is associated with increased Bad phosphorylation To further demonstrate whether rapamycin resistance involves Bad phosphorylation, rapamycin sensitive and rapamycin resistant lung cancer cells were generated as described previously. Phosphorylation levels of Bad at S112, S136 and S155 were compared in A549 P and A549 RR cells. Intriguingly, higher levels of Bad phosphorylation at S112 and S136 but not S155 in association with increased activities of ERK1/2 and Akt were observed in A549 RR cells as compared to A549 P cells. Importantly, sulforhodamine B colorimetric and colony formation assays reveal that A549 P is sensitive but A549 RR is insensitive to rapamycin. These provide strong evidence that rapamycin induced Bad phosphorylation at S112 and S136 contributes to rapamycin resistance in human lung cancer cells. To test whether the nonphosphorylatable mutations of Bad at S112 and S136 sites are sufficient to reverse rapamycin resistance, the non phosphorylatable Cyclopamine S112A/S136A mutant murine Bad as well as the WT murine Bad were transfected into A549 parental and rapamycin resistant A549 cells. After transfection, cells were treated with rapamycin for 48h. reveal that expression of the AA mutant Bad but not WT Bad reverses rapamycin resistance of A549 RR cells. Previous reports have demonstrated that murine Bad and human Bad have conserved structural homology and function. However, the number of amino acids in murine Bad and human Bad are different. Human Bad lacks a stretch of 42 amino acids as previously reported. This is consistent with our findings that the molecular weight of exogenous murine Bad is larger than the endogenous human Bad. Treatment of lung cancer cells with rapamycin in Bad accumulation in the cytosol, increased Bad/14 3 3 association and decreased Bad/Bcl XL binding Our indicate that rapamycin induces phosphorylation of Bad at S112 and S136.