• Title/Summary/Keyword: Spry2

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Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells

  • Ahn, Jun-Ho;Han, Byeal-I;Lee, Michael
    • Biomolecules & Therapeutics
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    • v.23 no.4
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    • pp.320-326
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    • 2015
  • The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

Spry2 does not directly modulate Raf-1 kinase activity in v-Ha-ras-transformed NIH 3T3 fibroblasts

  • Ahn, Jun-Ho;Eum, Ki-Hwan;Lee, Michael
    • BMB Reports
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    • v.43 no.3
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    • pp.205-211
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    • 2010
  • Sprouty (Spry) proteins have previously been suggested as negative regulators of the MAPK pathway through interaction with Raf-1. However, the molecular basis of this inhibition has not been elucidated. In this study, we used cells expressing FLAGtagged Raf-1 with point mutations at known phosphorylation sites to reveal that activation of Raf-1 mutants does not correlate with their degree of interaction with Spry2. The association of Raf-1 with Spry2 in intact cells was further corroborated by immunofluorescence colocalization. Additionally, there was no significant change observed in the strength of interaction between Raf-1 mutants and Spry2 after paclitaxel treatment despite differences in the activation levels of these mutants. Thus, our study provides the evidence that Spry2 does not directly regulate Raf-1 kinase activity, but instead acts as a scaffolding protein that assists interactions between Raf-1 kinase and its direct regulators.

Novel Function of Sprouty4 as a Regulator of Stemness and Differentiation of Embryonic Stem Cells

  • Lee, Jae-Young;Park, Sunghyun;Kim, Kwang-Soo;Ko, Jeong-Jae;Lee, Soohong;Kim, Keun Pil;Park, Kyung-Soon
    • Development and Reproduction
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    • v.20 no.2
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    • pp.149-155
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    • 2016
  • Sprouty (Spry) genes encode inhibitors of the receptor tyrosine kinase signaling cascade, which plays important roles in stem cells. However, the role of Spry4 in the stemness of embryonic stem cells has not been fully elucidated. Here, we used mouse embryonic stem cells (mESCs) as a model system to investigate the role of Spry4 in the stem cells. Suppression of Spry4 expression results in the decreases of cell proliferation, EB formation and stemness marker expression, suggesting that Spry4 activity is associated with stemness of mESCs. Teratoma assay showed that the cartilage maturation was facilitated in Spry4 knocked down mESCs. Our results suggest that Spry4 is an important regulator of the stemness and differentiation of mESCs.

Decreased Interaction of Raf-1 with Its Negative Regulator Spry2 as a Mechanism for Acquired Drug Resistance

  • Ahn, Jun-Ho;Kim, Yun-Ki;Lee, Michael
    • 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.

오갈피 나무속(Acanthopanax spry.) 식물의 번식에 관한 연구 -제1보 삽목시 생장조절성질이 발근에 미치는 영향-

  • 안상득;최은옥
    • Journal of Ginseng Research
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    • v.16 no.2
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    • pp.138-145
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    • 1992
  • Rooting test of 5 species plants in Acanthopanax genus was carried out to see the comparative effect of growth regulators NAA IBA and IAA. And the results are summarized as in the following compendia; (1) When hard and soft-wood cutting from new shoot of 5 species were taken and dipping treated with growth regulators such as NAA IBA and IAA at varied concentration levels their roofings were mostly more superior to the control that untreated. And also treatment of NAA and IBA among treated substances were more effective than IAA. (2) The effects on the rooting number and rate made a great difference according to species of Acanthopanax genus even if the same regulator was treated. (3) Decreasing effects of rooting by high concentration obstacle (especially at 2,000 rpm) of NAA and IBA in other species except A. sieboldianum were mostly serious. On the other hand, that in case of IAA treatment was not risen in most species except A. sessiliflorus.

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Biochemical and molecular characterization of a strain KA/S2 of Acnnthamoebc castellanii isolated from Korean soil (카스텔란니가시아메바(Acanthamoeba castellanii) 한국 토양분리주 KA/S2의 생화학적 및 분자생물학적 특성)

  • 정동일;공현희
    • Parasites, Hosts and Diseases
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    • v.34 no.1
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    • pp.79-86
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    • 1996
  • A strain, KA/S2, isolated from Korean soil and morphologically assigned to Acanthcmoebc cQsteLlcnii, was characterized by isoenzyme analysis , and total proteins profile, End mitochondrial (Mt) DNA restriction fragment length polymorphism (RFLP) , and compared with four reference strains assigned to the species (the authenitic Castellani, Neff, Ma, and Chang strains). It was found that four isoenzyme, total proteins, and Mt DNA RFLP patterns by eight restriction endonucleases of the strain KA/S2 were identical with those of the Neff strain, isolated from soil of California, USA. The Chang strain was unique in its morphology and total protein patterns. Interstrain polymorphisms of isoensyme profiles and Mt DNA RFLP patterns were observed among the Castellani, Neff, Ma, and Chang strains. Mt DNA RFLP was confirmed to be highly appropriate for the strain characterization and identification of Acnnthamoeba spry.

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Noonan syndrome and RASopathies: Clinical features, diagnosis and management

  • Lee, Beom Hee;Yoo, Han-Wook
    • Journal of Genetic Medicine
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    • v.16 no.1
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    • pp.1-9
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    • 2019
  • Noonan syndrome (NS) and NS-related disorders (cardio-facio-cutaneous syndrome, Costello syndrome, NS with multiple lentigines, or LEOPARD [lentigines, ECG conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth and sensory neural deafness] syndrome) are collectively named as RASopathies. Clinical presentations are similar, featured with typical facial features, short stature, intellectual disability, ectodermal abnormalities, congenital heart diseases, chest & skeletal deformity and delayed puberty. During past decades, molecular etiologies of RASopathies have been growingly discovered. The functional perturbations of the RAS-mitogen-activated protein kinase pathway are resulted from the mutation of more than 20 genes (PTPN11, SOS1, RAF1, SHOC2, BRAF, KRAS, NRAS, HRAS, MEK1, MEK2, CBL, SOS2, RIT, RRAS, RASA2, SPRY1, LZTR1, MAP3K8, MYST4, A2ML1, RRAS2). The PTPN11 (40-50%), SOS1 (10-20%), RAF1 (3-17%), and RIT1 (5-9%) mutations are common in NS patients. In this review, the constellation of overlapping clinical features of RASopathies will be described based on genotype as well as their differential diagnostic points and management.

Gene Expression Profiles in Cervical Cancer with Radiation Therapy Alone and Chemo-radiation Therapy (자궁경부암의 방사선치료 및 방사선항암화학 병용치료에 따른 유전자발현 조절양상)

  • Lee Kyu Chan;Kim Meyoung-kon;Kim Jooyoung;Hwang You Jin;Choi Myung Sun;Kim Chul Yong
    • Radiation Oncology Journal
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    • v.21 no.1
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    • pp.54-65
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    • 2003
  • Purpose : To analyze the gene expression Profiles of uterine ceulcal cancer, and its variation after radiation therapy, with or without concurrent chemotherapy, using a CDNA microarray. Materials and Methods :Sixteen patients, 8 with squamous ceil carcinomas of the uterine cervix, who were treated with radiation alone, and the other 8 treated w14h concurrent chemo-radiation, were Included in the study. Before the starling of the treatment, tumor biopsies were carried out, and the second time biopsies were peformed after a radiation dose of 16.2$\~$27 Gy. Three normal cervix tissues were used as a control group. The microarray experiments were peformed with 5 groups of the total RNAs extracted individually and then admixed as control, pre-radiation therapy alone, during-radiation therapy alone, pre-chemoradiation therapy, and during-chemoradlation therapy. The 33P-iabeled CDNAS were synthesized from the total RNAs of each group, by reverse transcription, and then they were hybridized to the CDNA microarray membrane. The gene expression of each microarrays was captured by the intensity of each spot produced by the radioactive isotopes. The pixels per spot were counted with an Arrayguage, and were exported to Microsoft Excel The data were normalized by the Z transformation, and the comparisons were peformed on the Z-ratio values calculated. Results : The expressions of 15 genes, including integrin linked kinase (ILK), CDC28 protein kinase 2, Spry 2, and ERK 3, were increased with the Z-ratio values of over 2.0 for the cervix cancer tissues compared to those for the normal controls. Those genes were involved In cell growth and proliferation, cell cycle control, or signal transduction. The expressions of the other 6 genes, Including G protein coupled receptor kinase 5, were decreased with the Z-ratio values of below -2.0. After the radiation thorapy, most of the genes, with a previously Increase expressions, represented the decreased expression profiles, and the genes, with the Z-ratio values of over 2.0, were cyclic nucleotlde gated channel and 3 Expressed sequence tags (EST). In the concurrent chemo-radiation group, the genes involved in cell growth and proliferation, cell cycle control, and signal transduction were shown to have increased expressions compared to the radiation therapy alone group. The expressions of genes involved in anglogenesis (angiopoietln-2), immune reactions (formyl peptide receptor-iike 1), and DNA repair (CAMP phosphodiesterase) were increased, however, the expression of gene involved In apoptosls (death associated protein kinase) was decreased. Conclusion : The different kinds of genes involved in the development and progression of cervical cancer were identified with the CDNA microarray, and the proposed theory is that the proliferation signal stalls with ILK, and is amplified with Spry 2 and MAPK signaling, and the cellular mitoses are Increased with the increased expression oi Cdc 2 and cell division kinases. After the radiation therapy, the expression profiles demonstrated 4he evidence of the decreased cancer cell proliferation. There was no sigificant difference in the morphological findings of cell death between the radiation therapy aione and the chemo-radiation groups In the second time biopsy specimen, however, the gene expression profiles were markedly different, and the mechanism at the molecular level needs further study.

Studies on the Relationship Between the Presence of Plasmids and the Tumor cell's Cytotoxicity Shown by the Streptomyces spp (Streptomyces속 야생균주들이 생산하는 세포독성물질과 plasmid와의 연관성에 관한 연구)

  • Kim, Mi-Yong;Shin, Suck-Woo;Choi, Boung-Don;Ryeom, Kon
    • Biomolecules & Therapeutics
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    • v.3 no.2
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    • pp.154-158
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    • 1995
  • We isolated Streptomyces spry. from Korean soil, which showed high cytotoxicity against tumor cell lines, L1210 and P388Dl. Among 30 strains, three strains (DKM 104, DKM 128, DKM 409) were appeared to possess plasmid. Strain DKM 104 and DKM 128 had two CCC(Covalently Closed Circular) form plasmid, about 20 Kb in size and about 1 Kb compared with λ Hind III DNA size marker. And strain DKM 409 had three plasmids, among which two plasmic were CCC form about 20 Kb and about 20 Kb compared with same size marker. To find out whether plasmid involved in production of antitumor agent or not, we performed to curing experiment. Comparing cytotoxicity between culture filtrate of plasmid-containing strains and cured strains, we knew that only the cytotoxic activity of the strain DKM 128 was involved in plasmid.

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Close relatedness of Acanthomoeba pintulosa with Accnthcmoebc palestinensis based on isoenzyme profiles and rDNA PCR-RFLP patterns (Acanthamoeba pustulosa와 A. palestinensis의 동위효소 및 rDNA PCR-RFLP 양상의 유사성)

  • 김영호;옥미선
    • Parasites, Hosts and Diseases
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    • v.34 no.4
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    • pp.259-266
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    • 1996
  • The taxonomic validity of morphological group III Accnthamoeba app. is uncertain. In the present study. six type strains of group III Aconthamoeba spry. , A. culbertsoni, A. heniyi, A. pustulosc, A. palestinensis, A. royrebn and A. lenticulnto were subjected for the evaluation or their taxonomic validity by comparison of the isoeneyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment length polymorphism (RFLP) . and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between the species. The type strains of A. pclestinensls and A. pustulosc showed almost identical patterns of isoenrymes and rDNA PCR-RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenxymes and rDNA PCR- RFLP. It is likely that A. pustuLosc is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.

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