• Biomolecules & Therapeutics
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• v.21 no.2
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• pp.114-120
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• 2013

Most patients with mutant B-Raf melanomas respond to inhibitors of oncogenic B-Raf but resistance eventually emerges. To better understand the mechanisms that determine the long-term responses of mutant B-Raf melanoma cells to B-Raf inhibitor, we used chronic selection to establish B-Raf (V600E) melanoma clones with acquired resistance to the new oncogenic B-Raf inhibitor UI-152. Whereas the parental A375P cells were highly sensitive to UI-152 ($IC_{50}$ < $0.5{\mu}M$), the resistant sub-line (A375P/Mdr) displayed strong resistance to UI-152 ($IC_{50}$ < $20{\mu}M$). Immunofluorescence analysis indicated the absence of an increase in the levels of P-glycoprotein multidrug resistance (MDR) transporter in A375P/Mdr cells, suggesting that resistance was not attributable to P-glycoprotein overexpression. In UI-152-sensitive A375P cells, the anti-proliferative activity of UI-152 appeared to be due to cell-cycle arrest at $G_0/G_1$ with the induction of apoptosis. However, we found that A375P/Mdr cells were resistant to the apoptosis induced by UI-152. Interestingly, UI-152 preferentially induced autophagy in A375P/Mdr cells but not in A375P cells, as determined by GFP-LC3 puncta/cell counts. Further, autophagy inhibition with 3-methyladenine (3-MA) partially augmented growth inhibition of A375P/Mdr cells by UI-152, which implies that a high level of autophagy may protect UI-152-treated cells from undergoing growth inhibition. Together, our data implicate high rates of autophagy as a key mechanism of acquired resistance to the oncogenic B-Raf inhibitor, in support of clinical studies in which combination therapy with autophagy targeted drugs is being designed to overcome resistance.

• Molecules and Cells
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• v.42 no.9
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• pp.646-660
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• 2019

Abscisic acid (ABA) is a phytohormone essential for seed development and seedling growth under unfavorable environmental conditions. The signaling pathway leading to ABA response has been established, but relatively little is known about the functional regulation of the constituent signaling components. Here, we present several lines of evidence that Arabidopsis Raf-like kinase Raf10 modulates the core ABA signaling downstream of signal perception step. In particular, Raf10 phosphorylates subclass III SnRK2s (SnRK2.2, SnRK2.3, and SnRK2.6), which are key positive regulators, and our study focused on SnRK2.3 indicates that Raf10 enhances its kinase activity and may facilitate its release from negative regulators. Raf10 also phosphorylates transcription factors (ABI5, ABF2, and ABI3) critical for ABA-regulted gene expression. Furthermore, Raf10 was found to be essential for the in vivo functions of SnRK2s and ABI5. Collectively, our data demonstrate that Raf10 is a novel regulatory component of core ABA signaling.

• Journal of Life Science
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• v.9 no.2
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• pp.52-56
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• 1999

The Raf, a cytoplasmic serine/thereonine protein kinase, acts as an important mediator of signals involving cell proliferation, differentiation and development. Multiple regulatory elements should participate in the expression of D-raf, Drosophila homolog of human c-raf-1. In order to search regulatory factors involved in the D-raf promoter activation, we accomplished the yeast one-hybrid screening using D-raf promoter region from bp-330 to -309 with respect to the transcription initiation site as bait. After screening, sixteen independent positive clones of ${\beta}$-galactosidase activties were identified and sequenced. Two clones having 94-98% identity with daughterless and one clone having 93% identity with escargot by Blast search among these clones were screened.

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.11b
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• pp.136-136
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• 2002

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance and apoptosis induced by paclitaxel. In the present study, paclitaxel sensitivity, Raf-1 activity and MAPKs activation were compared in 2 cell lines: parental human breast cancer cells and its drug resistant variant (MCF-7/Adr) cells.(omitted)

• Korean Journal of Plant Taxonomy
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• v.44 no.3
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• pp.181-187
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• 2014

The taxonomic status of a new forma, Opuntia humifusa (Raf.) Raf. f. jeollaensis E. J. Kim & S. S. Whang, and the taxonomic relationships of three Korean cladodes were studied based on DNA sequences of three genes. The new forma O. humifusa f. jeollaensis (Wanggasi-Chunnyuncho) is simlilar to O. humifusa (Chunnyuncho), but differ in having both flower with the reddish inner center, and strong and many 3 cm long spines. Molecular studies showed that the new forma grouped into Macrocentra series instead of Humifusa series which included Korean O. humifusa. We designated the new taxa firstly studied here as a new forma, because the taxa have been mainly cultivated in farmlands in Jeolla-do area rather than distributed in nature. The taxonomic relationships of three Korean cladodes are discussed in detail.

• Biomolecules & Therapeutics
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• v.19 no.2
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• pp.174-180
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• 2011

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance to paclitaxel in v-H-ras transformed NIH 3T3 fibroblasts (Ras-NIH 3T3). We established a multidrug-resistant cell line (Ras-NIH 3T3/Mdr) from Ras-NIH 3T3 cells by stepwise increases in paclitaxel. Drug sensitivity assays indicated that the $IC_{50}$ value for drug-resistant Ras-NIH 3T3/Mdr cells was more than 1 ${\mu}M$ paclitaxel, 10- or more-fold higher than for the parental Ras-NIH 3T3 cells. Western blot and RT-PCR analysis showed that the drug efflux pump a P-glycoprotein were highly expressed in Ras-NIH 3T3/Mdr cells, while not being detectable in Ras-NIH 3T3 cells. Additionally, verapamil, which appears to inhibit drug efflux by acting as a substrate for P-glycoprotein, completely reversed resistance to paclitaxel in Ras-NIH 3T3/Mdr cell line, indicating that resistance to paclitaxel is associated with overexpression of the multidrug resistance gene. Interestingly, Ras-NIH 3T3/Mdr cells have higher basal Raf-1 activity compared to Ras-NIH 3T3 cells. Unexpectedly, however, the colocalization of Raf-1 and its negative regulator Spry2 was less observed in cytoplasm of Ras-NIH 3T3/Mdr cells due to translocation of Spry2 around the nucleus in the perinuclear zone, implying that Raf-1 may be released from negative feedback inhibition by interacting with Spry2. We also showed that shRNA-mediated knockdown of Raf-1 caused a moderate increase in cell susceptibility to paclitaxel. Thus, the results presented here suggest that a Raf-1-dependent pathway plays an important role in the development of acquired drug-resistance.

• Animal cells and systems
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• v.1 no.1
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• pp.151-155
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• 1997

Promoter of the Drosophila proliferating cell nuclear antigen (PCNA) gene contains DRE (Drosophila DNA replication-related element) required for the high level expression of replication-related genes. Recently, we found that promoter region of the D-raf (a Drosophila homolog of the human c-raf-1) contains two sequences homologous to the DRE and demonstrated the DRE/DREF (DRE-binding factor) involvement in regulation of the D-raf gene. In this study, using ursolic acid (UA), a pentacyclic triterpene acid reported to possess antitumor activities, we examined effects of UA on proliferation of the Drosophila cultured Kc cells and on expression of the PCNA and D-raf genes. UA showed an inhibitory effect on proliferation of the Kc cells in a concentration-dependent manner in DNA content assays and [3H]thymidine incorporation assays. The IC50 value of anti-proliferative effects of UA in DNA content assays was about 7.5uM. UA showed inhibitory effects on expression of the PCNA as well as on that of the D-raf, which were examined with the reporter plasmic p5'-168DPCNACAT or p5'-878DrafCAT, respectively. The results obtained in the present study suggest that expression of the PCNA and D-raf genes is coordinately regulated in at least UA-treated Kc cells and that down-regulation of expression of the PCNA and D-raf genes might be related with the antitumor activities of UA.

• Macromolecular Research
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• v.9 no.4
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• pp.228-237
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• 2001

The evolution of conformational constraints in bisphenol-A polycarbonate (BAPC) upon quiescent bulk crystallization was quantitatively analyzed from calorimetric study employing a rigid amorphous fraction (RAF) as an indicator of the level of conformational constraints. From the correlation between corrected crystallinity (X$\sub$c/) and total rigid fraction (f$\sub$r/), it was found that, regardless of molar mass distribution and thermal treatment conditions, semicrystalline BAPC always exhibits greater f$\sub$r/ than X$\sub$c/ maintaining a quantitative relationship of f$\sub$r/〓2X$\sub$c/ in the range of 0.0 $\sub$c/< 0.4. This directly indicates the evolution of approximately the same amount of RAF as X$\sub$c/, (i.e., RAF〓X$\sub$c/) upon bulk crystallization of BAPC. It was also found that T$\sub$g/ per se and T$\sub$g/ broadening enhance as RAF increases, and there appears to be a critical level of RAF (>0.2) needed to initiate significant changes in both quantities.

• Korean Journal of Food Science and Technology
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• v.52 no.4
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• pp.385-395
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• 2020

This study examined the effect of 3,4-dihydroxytoluene (DHT) on UVB-induced photoaging and determined its molecular mechanisms, using HaCaT human keratinocytes and SKH-1 hairless mice. DHT suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression in HaCaT cells. In vivo data from mouse skin supported that DHT decreased UVB-induced wrinkle formation, epidermal thickness, and matrix metalloproteinase-13 (MMP-13) expression. DHT appeared to exert its anti-aging effects by suppressing UVB-induced Raf-1 kinase activity and subsequent attenuation of UVB-induced phosphorylation of MEK, ERK, and p90RSK in HaCaT cells. In vitro and in vivo pull-down assays revealed that DHT bound with Raf-1 in ATP-noncompetitive manner. Overall, DHT appears to anti-photoaging effects in vitro and in vivo through the suppression of Raf-1 kinase activity and may have potential as a treatment for the prevention of skin aging.

• Journal of Life Science
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• v.26 no.1
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• pp.101-108
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• 2016

The aim of this study was to evaluate the relationships between the expression of Heat shock protein70 (HSP70), Raf-1 and Bcl-2-associated athanogene-1 (BAG1) protein in diffuse type gastric carcinoma and examine association of HSP70, Raf-1 and BAG1 expression with various clinic-pathological factors and survival. Heat shock protein70 is induced in the cells in response to various stress conditions, including carcinogens. Overexpression of heat shock protein 70 has been observed in many types of cancer. The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. Overexpression of BAG1 protein has been documented in some type of human cancer. BAG1 has been reported to interact with protein involved with a variety of signal pathway, and regulation of cell differentiation, survival and apoptosis. These interaction partners include HSP70 and Raf-1. The percentage of tumors exhibiting HSP70 positivity was significantly in cases of positive lymph node metastasis (64.9%) compared to cases without lymph node metastasis (35.1%, p=0.007). HS70 expression was correlated with pathological N-stage (p=0.006). Expression of BAG1 was detected in the majority of diffuse type gastric carcinoma tissues (71.7%), especially in younger patients (80% vs 52.6%, p=0.035). Furthermore BAG1 expression was correlated with tumor size (p=0.020). Raf-1 expression was found to be significantly associated with tumor size (p=0.005). The result indicate that HSP70 was significantly correlated the progression of diffuse type gastric cancer. Expression of BAG1 and Raf-1 may be used as diagnostic markers for gastric carcinoma.