• Molecules and Cells
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• v.25 no.2
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• pp.184-195
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• 2008

We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP ($c-FLIP_L$), but not the short form ($c-FLIP_S$), enhanced adhesion and motility. Downregulation of $c-FLIP_L$ with siRNA decreased phosphorylation of FAK and ERK, while overexpression of $c-FLIP_L$ increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed $c-FLIP_L$-promoted motility. Inhibition of ROCK by Y27632 suppressed the $c-FLIP_L$-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of $c-FLIP_L$ increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced $c-FLIP_L$- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the $c-FLIP_L$-promoted cell motility. We conclude that $c-FLIP_L$ promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.6
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• pp.2251-2256
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• 2015

To study effects of cellular FLICE (FADD-like IL-$1{\beta}$-converting enzyme)-inhibitory protein (c-FLIP) inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screened from the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantly suppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the control cells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAILinduced cell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs) AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs. Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA and rocaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a good target for anti-cancer therapy.

• Bulletin of the Korean Chemical Society
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• v.27 no.1
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• pp.87-92
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• 2006

Fas-associated death domain protein (FADD) recruits and activates procaspase-8 through interactions between the death effector domains of these two proteins. Cellular FLICE-inhibitory protein (c-FLIP) was identified as a molecule with sequence homology to caspase-8. It has been postulated that c-FLIP prevents formation of the competent death-inducing signaling complex in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. However, the interaction of FADD and $c-FLIP_s$ (short form) in apoptosis signaling has been controversially discussed. We show the purification and the characterization of human full-length FADD and $c-FLIP_s$ expressed in Escherichia coli. The purified FADD and $c-FLIP_s$ are shown as homogeneity, respectively, in SDS-PAGE analysis and light-scattering measurements. The folding properties of the $\alpha$-helical structure of FADD and the super-secondary structure of $c-FLIP_s$ proteins were characterized by circular dichroism spectroscopy. Furthermore, we report here a series of biochemical and biophysical data for FADD-$c-FLIP_s$ binding in vitro. The binding of both FADD and $c-FLIP_s$ proteins was detected by BIAcore biosensor, fluorescence measurement, and size-exclusion column (SEC).

• BMB Reports
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• v.47 no.9
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• pp.488-493
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• 2014

Adaptor protein FADD forms the death inducing signaling complex (DISC) by recruiting the initiating caspases-8 and -10 through homotypic death effector domain (DED) interactions. Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of death ligand-induced apoptosis downstream of death receptors, and FADD competes with procaspase-8/10 for recruitment for DISC. However, the mechanism of action of FADD and c-FLIP proteins remain poorly understood at the molecular level. In this study, we provide evidence indicating that the death effector domain (DED) of FADD interacts directly with the death effector domain of human c-FLIP. In addition, we use homology modeling to develop a molecular docking model of FADD and c-FLIP proteins. We also find that four structure-based mutants (E80A, L84A, K169A and Y171A) of c-FLIP DEDs disturb the interaction with FADD DED, and that these mutations lower the stability of the c-FLIP DED.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.401-409
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• 2010

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

• Journal of the Microelectronics and Packaging Society
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• v.15 no.4
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• pp.9-15
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• 2008

Microstructure and contact resistance of the Au-Sn solder joints were characterized after flip-chip bonding of the Au/Sn bumps processed by successive electrodeposition of Au and Sn. Microstructure of the Au-Sn solder joints, formed by flip-chip bonding at $285^{\circ}C$ for 30 sec, was composed of the $Au_5Sn$+AuSn lamellar structure. The interlamellar spacing of the $Au_5Sn$+AuSn structure increased by reflowing at $310^{\circ}C$ for 3 min after flip-chip bonding. While the Au-Sn solder joints formed by flip-chip bonding at $285^{\circ}C$ for 30 sec exhibited an average contact resistance of 15.6 $m{\Omega}$/bump, the Au-Sn solder joints reflowed at $310^{\circ}C$ for 3 min after flip-chip bonding possessed an average contact resistance of 15.0 $m{\Omega}$/bump.

• Proceedings of the International Microelectronics And Packaging Society Conference
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• 2003.09a
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• pp.253-257
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• 2003

The low temperature flip chip technique is applied to the package of the temperature-sensitive devices for LCD systems and image sensors since the high temperature process degrades the polymer materials in their devices. We will introduce the various low temperature flip chip bonding techniques; a conventional flip chip technique using eutectic Bi-Sn (mp: $138^{\circ}C$) or eutectic In-Ag (mp: $141^{\circ}C$) solders, a direct bump-to-bump bonding technique using solder bumps, and a low temperature bonding technique using low temperature solder pads.

• Journal of the Microelectronics and Packaging Society
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• v.17 no.3
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• pp.27-32
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• 2010

For the flip chip joints processed using Cu pillar bumps and Sn pads, thermal cycling and high temperature storage reliabilities were examined as a function of the Sn pad height. With increasing the height of the Sn pad, which composed of the flip chip joint, from 5 ${\mu}m$ to 30 ${\mu}m$, the contact resistance of the flip chip joint decreased from 31.7 $m{\Omega}$ to 13.8 $m{\Omega}$. Even after thermal cycles of 1000 times ranging from $-45^{\circ}C$ to $125^{\circ}C$, the Cu pillar flip chip joints exhibited the contact resistance increment below 12% and the shear failure forces similar to those before the thermal cycling test. The contact resistance increment of the Cu pillar flip chip joints was maintained below 20% after 1000 hours storage at $125^{\circ}C$.

• Nuclear Engineering and Technology
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• v.11 no.1
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• pp.29-45
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• 1979

In this paper. nuclear characteristics of TRIGA Mark-III has been analyzed in detail for six different fuel options. Presently, 70 w/o enriched FLIP fuels are adopted for TRIGA core to improve fuel lifetime. However, such highly enriched fuels are not easily obtained due to nonproliferation treaty. This research examines the possible substitution for FLIP fuels with high density fuels without reducing the nuclear performance. This work will provide long-time plan for TRIGA operation.

• Journal of the Korean Institute of Telematics and Electronics C
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• v.35C no.1
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• pp.10-14
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• 1998

In this paper a double edge triggered (DET) filp-flop is proposed which changes its output state at both the positive and the negative edge transitions of the triggering input. DET filp-flop has advantages in terms of speed and power dissipation over single edge triggered (SET) filp-flop has proposed DET flip-flop needs only 12 MOS transistors and can operate at clock speed of 500 MHz. Also, the power dissipation has decreased about 33% in comparison to SET flip-flop.