[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-alpha-induced apoptosis, we isolated and characterized a novel TNF-alpha-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-alpha and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-alpha in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-alpha-induced apoptosis signaling. In UA cells, TNF-alpha-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1beta converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria.
[Show abstract][Hide abstract] ABSTRACT: Human monocytic leukemia U937 cells readily undergo apoptosis when they are treated with TNF-alpha, anti-Fas antibody and anticancer drugs such as etoposide and Ara-C. To study the mechanism of apoptosis, we developed a novel apoptosis-resistant variant, UC, from U937 cells. The UC cells showed resistance to apoptosis induced by TNF-alpha, anti-Fas antibody, etoposide and Ara-C. Somatic cell hybridization between U937 and UC showed that apoptosis-resistance to TNF-alpha in UC was genetically recessive and resistance to etoposide was dominant, suggesting that UC has at least two different mutations functionally involved in apoptosis. Mechanistic analysis revealed that UC cells expressed reduced amounts of c-Myc. Transfection of the c-myc gene into UC cells restored the sensitivity of the cells to undergo apoptosis induced by TNF-alpha and anti-Fas, which attributes apoptosis-resistance in this circumstance to the reduced expression of c-Myc. On the other hand, c-myc transfection into UC cells could not restore their sensitivity to etoposide- and Ara-C-induced apoptosis, arguing against the role of c-myc in chemotherapy-induced apoptosis. However, treating the parental U937 cells with antisense oligonucleotides designed to reduce c-Myc expression rendered the cells resistant to etoposide-induced as well as to TNF-alpha-induced apoptosis. These results indicate that the reduced expression of c-Myc in UC is strongly associated with the resistance to etoposide-induced apoptosis. Our finding that c-myc transfection into UC could not restore the sensitivity to etoposide-induced apoptosis, suggests UC could have a second mutation that confers resistance to etoposide-induced apoptosis in a genetically dominant manner. Taken together, our present results indicate that c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis.
[Show abstract][Hide abstract] ABSTRACT: P-glycoprotein, a multidrug transporter protein, exists in the brain capillary endothelium. To study the function of P-glycoprotein in brain capillary endothelium as a barrier against cyclosporin A, we examined the interaction of cyclosporin A with P-glycoprotein expressed in cultured brain capillary endothelial cells (MBEC4). P-glycoprotein of MBEC4 specifically bound [125I]iodoaryl azidoprazosin, and the binding was inhibited by cyclosporin A and vincristine. Intracellular accumulation of cyclosporin A in MBEC4 was about one-third the amount accumulated in mouse aortic endothelial cells (MAEC3), a cell line that did not express P-glycoprotein. The reduced accumulation of cyclosporin A in MBEC4 was increased by verapamil, a competitive inhibitor of transport function of P-glycoprotein. Cyclosporin A was preferentially transported from basal to apical side when the cell monolayer of MBEC4 was formed; however this transendothelial transport was not observed across cell monolayer of MAEC3. Verapamil inhibited the transendothelial transport of cyclosporin A across the MBEC4 monolayer. Thus P-glycoprotein in brain capillary endothelium could transport cyclosporin A across the endothelium from the basal to the apical side. These observations suggest that P-glycoprotein is involved in the complex function of the blood-brain barrier as a secretory detoxifying transporter of cyclosporin A.