Molecular mechanisms of TNF-α-induced ceramide formation in human glioma cells:P53-mediated oxidant stress-dependent and -independent pathways

Abstract

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-a-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-a stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O 2 ÀK ) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-a and lacked O 2 ÀK generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-a by accumulation of O 2 ÀK . z-IETD-fmk (benzyloxycarbonyl-Ile-GluThr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-a-induced ceramide formation through both SMases as well as O 2 ÀK generation. Caspase-8 was processed by TNF-a regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-a-induced apoptosis of human glioma cells.

HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes

“…Construction of the destination vector, pDEST-CL-SI-MSCVpuro, for retroviral expression of shRNA, pCL-SI-MSCVpuro-ErbB2R-shRNA, -HPV16E6-shRNA, -p53-shRNA and -E6AP-shRNA was carried out by a previously described method (Sawada et al, 2004). The shRNA targeted sequences were as follows: HPV16E6: GTATGGAACAACATTAGAA, E6AP: 5 0 -GAAATCTAGT GAATGATGA-3 0 , p53 (18) and ErbB2 (Yang et al, 2004) as described previously.…”

Ceramide: A common pathway for atherosclerosis?

“…Furthermore, they found that, indeed, TNF-α caused a rapid increase of superoxide free radical within 2 minutes, but the effect was not sustained and dissipated after 60 minutes [24,25]. TNF-α can stimulate ceramide formation via the activation of both neutral and acid SMases, accompanied by ROS such as superoxide anion production [26]. TNF-α initiates the pathway through TNFR1 (55-kDa receptor) leading to phospholipase A2 activation, generation of arachidonic acid, and subsequent activation of SMase [27].…”

Creation of tumorigenic human endometrial epithelial cells with intact chromosomes by introducing defined genetic elements

“…Coding sequences of the oncogenic mutant K-ras (Kras-G12V) and an activated form of Akt with a src myristoylation sequence (myrAkt) (Kohn et al, 1996) were first recombined into pDONR201 or pDONR221 (Invitrogen, Carlsbad, CA, USA) by performing the BP reaction according to the manufacturer's instruction, and were then recombined into the destination vector, pDEST-CMSCVpuro, by performing the LR reaction (Invitrogen, Carlsbad, CA, USA) to generate pCMSCVpuro-K-ras* and pCMSCVpuro-myr-Akt. To generate a retrovirus vector expressing PTEN-specific shRNA, the annealed oligos (5 0 gatccccGACATTATGACACCGCC AAttcaagagaTTGGCGGTGTCATAATGTCtttttggaaa3 0 and 5 0 agcttttccaaaaaGACATTATGACACCGCCAAtctcttgaaTT GGCGGTGTCATAATGTCggg3 0 ) were replaced with the stuffer of pENTR221-H1R-stuffer (Sawada et al, 2004). The shRNA expression cassette was recombined into pDEST-CL-SI-MSCVpuro (previously called pSI-CMSCVpuro-DEST) (Sawada et al, 2004), by performing the LR reaction (Invitrogen) according to the manufacturer's instruction to generate pCL-SI-MSCVpuro-H1R-PTENRi8.…”

“…To generate a retrovirus vector expressing PTEN-specific shRNA, the annealed oligos (5 0 gatccccGACATTATGACACCGCC AAttcaagagaTTGGCGGTGTCATAATGTCtttttggaaa3 0 and 5 0 agcttttccaaaaaGACATTATGACACCGCCAAtctcttgaaTT GGCGGTGTCATAATGTCggg3 0 ) were replaced with the stuffer of pENTR221-H1R-stuffer (Sawada et al, 2004). The shRNA expression cassette was recombined into pDEST-CL-SI-MSCVpuro (previously called pSI-CMSCVpuro-DEST) (Sawada et al, 2004), by performing the LR reaction (Invitrogen) according to the manufacturer's instruction to generate pCL-SI-MSCVpuro-H1R-PTENRi8. The backbone vector, pCL-SI-MSCVpuro-H1R, was used as a control (Sawada et al, 2004).…”

“…The shRNA expression cassette was recombined into pDEST-CL-SI-MSCVpuro (previously called pSI-CMSCVpuro-DEST) (Sawada et al, 2004), by performing the LR reaction (Invitrogen) according to the manufacturer's instruction to generate pCL-SI-MSCVpuro-H1R-PTENRi8. The backbone vector, pCL-SI-MSCVpuro-H1R, was used as a control (Sawada et al, 2004). Production of recombinant retroviruses is described in detail elsewhere (Kyo et al, 2003).…”