• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.104-104
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• 2001

Ras는 세포의 성장과 분화 등 여러 필수적인 세포기능에 없어서는 안될 중요한 역할을 담당하며 Ras가 mutation되면 암 등의 치명적인 결과를 초래한다. Ras 발현은 유방암에서 tumor aggressiveness의 지표로 간주되고 있으며 유방세포의 침습성과 연관이 있다고 알려져 있으므로 ras가 전이과정에 미치는 영향에 관한 연구는 중요한 의미를 갖는다. 본 연구의 선행연구결과, H-ras와 N-ras 모두 transformed phenotype을 나타내지만 H-ras 만이 암전이에 있어서 중요한 침윤성을 유도하는 것을 밝혔다. 이 결과는 MCF10A 세포에서 H-ras와 N-ras에 의한 신호전달경로가 각각 다른 생물학적 전이활성을 나타냄을 시사한다. 세포의 이동성은 침습성에 있어서 결정적인 역할을 하므로, 본 연구에서 H-ras와 N-ras로 형질전환된 MCF10A세포에서 이동성을 시험한 결과, 세포의 이동성이 N-ras가 아닌 H-ras MCF10A 세포에서만 크게 증가된다는 것을 보았다. 이는 침습성을 나타내는 H-ras가 세포의 이동성을 증가시키는데 작용한다는 것을 말한다. H-ras에 의해 유도된 침습성과 이동성에 대한 분자적 기전에 관하여 연구하기 위하여 H-ras MCF10A와 N-ras MCF10A 세포에서 Ras의 downstream effector들, 특히 mitogen-activated protein kinases(MAPKinases)들인 JNK1, ERK, p38의 활성화를 살펴본 결과 p38 MAPKinase가 H-ras MCF10A 세포에서 현저하게 활성화됨을 보았다. p38 MAPKinase 저해제인 SB203580를 처리하던지 dominant negative p38 (DN p38) transfectant로 p38을 불활성화시켰을 때 세포침습성 및 이동성이 저해되는 결과를 얻었다. SB203580 처리한 H-ras MCF10A 세포에서 전이에 관여하는 효소인 MMP-2 분비가 감소되었다. H-ras에 의해 유도된 침습성과 이동성은 DN JNK1 transfectant에서는 변화가 없었으나 DN MEK transfectants에서는 유의성있게 감소되었다. 이상의 결과를 종합하면, MCF10A 세포의 침윤성과 이동성에는 p38 MAPKinase 활성이 중심적인 역할을 하며, JNK 활성은 영향을 미치지 않고, ERK-1/2 활성은 충분하지는 않으나 필요하다는 것을 알 수 있었다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.103-103
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• 2001

인간유방상피세포에서 H-ras가 침윤성과 세포 이동성을 유도한다는 것을 이 전연구에서 밝혔다. Phosphatidylinositol 3-kinase(PI3K)는 세포 이동성에서 중요한 역할을 하는 것으로 보고되고 있다. 본 연구에서 인간유방상피세포인 MCF10A에서 H-ras에 의해 유도된 침윤성에 PI3K가 어떠한 영향을 미치는지 살펴보고자 하였다. PI3K의 활성은 PI3K의 downstream molecule인 Akt의 인산화를 Western blot으로 확인하였다. Akt는 MCF10A, H-ras, N-ras MCF10A 세포에서 같은 정도로 발현되는 반면, 인산화된 Akt는 MCF10A 세포에 비해 H-ras MCF10A 세포와 N-ras MCF10A 세포에서 현저히 높게 나타났다. 이상의 결과로서 H-ras, N-ras 둘 다 PI3K를 활성화시키며, 침윤성과 세포이동성이 없는 N-ras MCF10A 세포에서도 PI3K가 활성화되었으므로, PI3K의 활성은 세포침윤성과 이동성을 유도하는데에 있어서 충분하지는 않음을 말해준다. PI3K의 저해제인 LY294002와 wortmannin을 세포에 처리하였을 때 세포침윤성과 이동성이 유의성 있게 감소되었다. 이상의 결과는 MCF10A 세포의 침윤성과 이동설에 있어서 PI3K의 활성이 충분하지는 않지만 반드시 필요하다는 것을 알 수 있었다.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.267-276
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• 2020

T In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-Ras^V12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-Ras^V12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-Ras^V12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-Ras^V12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-Ras^V12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that Ras^V12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.26 no.6
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• pp.581-590
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• 2000

Alterations in the cellular genome affecting the expression or function of genes controlling cell growth and differentiation are considered to be the main cause of cancer. Over 30 oncogenes can be activated by insertional mutagenesis, single point mutations, chromosomal translocations and gene amplification. The ras oncogenes have been detected in $15{\sim}20%$ of human tumors that include some of the most common forms of human neoplasia and are known to acquire their transforming properties by single point mutations in two domains of their coding sequences, most commonly in codons 12 and 61. The ras gene family consists of three functional genes, N-ras, K-ras and H-ras which encode highly similar proteins of 188 or 189 amino acid residues generically known as P21. ras proteins have been shown to bind GTP and GTP, and possess intrinsic GTPase activity. Experimental study was performed to observe the mutational change of the ras gene family and apply the results to the clinical activity. 36 Golden Syrian Hamster each weighing $60{\sim}80g$ were used and painted with 0.5% DMBA by 3 times weekly on the right buccal cheek(experimental side) for 6, 8, 10, 12, 14 and 16 weeks. Left buccal cheek (control side) was treated with mineral oil as the same manner of the right side. The hamsters were sacrificed on the 6, 8, 10, 12, 14 & 16 weeks. Normal and tumor tissues from paraffin block were completely dissected by microdissection and DNA from both tissue were isolated by proteinase K/phenol/chloroform extraction. Segments of the K-ras and H-ras gene were amplified by PCR using the oligonucleotide primers corresponding to the homologous region (codon 12 and 61) of the hamster gene, and then confirmational change of ras genes was observed by SSCP and autosequencing analysis. The results were as follows : 1. Malignant lesion could be found in the experimental side from the experimental six weeks. 2. One hamster among six showed point mutation of the H-ras codon 12($G{\rightarrow}A$ transition) at the experimental 10 and 14 weeks. 3. One of six at 6 weeks, two of six at 8 weeks and one of six at 12 weeks revealed the confirmational change of the H-ras codon 61($A{\rightarrow}T$ transversion). 4. The incidence of point mutation of H-ras codon 12 and 61 were 5.5%(2 of 36) and 11%(4 of 36) respectively. 5. Point mutation of the K-ras could not be seen during the whole experimental period. Form the above results, these findings strongly support the concept that H-ras oncogenes may have the influence of the DMBA induced carcinoma of hamster buccal pouch.

• BMB Reports
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• v.47 no.12
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• pp.685-690
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• 2014

The Ras/Raf/MEK/Erk signaling pathway is important for regulation of cell growth, proliferation, differentiation, survival, and apoptosis in response to a variety of extracellular stimuli. Lack of Erk MAPK activation is observed in several cancer cells despite active activation of Ras. However, little is known about the modulation of Erk1/2 activity by active Ras. Here, we show that overexpression of active H-Ras (H-RasG12R) in NIH3T3 fibroblasts impaired FGF2-induced Erk1/2 phosphorylation, as compared to wild-type cells. Northern blot analysis revealed that prolonged expression of active Ras increased MAP kinase phosphatase 3 (MKP3) mRNA expression, a negative regulator of Erk MAPK. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway abrogated active Ras-induced up-regulation of MKP3 expression, leading to the rescue of Erk1/2 phosphorylation. Our results demonstrated that the Ras/Raf/MEK/Erk signaling cascade is negatively regulated by the PI3K/Aktdependent transcriptional activation of the MKP3 gene.

• Biomolecules & Therapeutics
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• v.6 no.4
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• pp.345-350
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• 1998

The adverse effects of ethanol on cell proliferation have been described for a variety of tissues and cells. In the present study, we investigated whether chronic ethanol intoxication impairs the cell proliferation and DNA synthesis induced by oncogenic $H-ras^{V12}$ - and $v-K-ras^{V12}$-transformed cells. Ethanol treatment inhibited the cell proliferation and the DNA synthesis of control parental fibroblasts in a time- and dose-dependent manner. In contrast, ethanol did not suppress the proliferation of either oncogenic $H-ras^{V12}$ - or $v-K-ras^{V12}$ -transformed fibroblasts. Microinjection of oncogenic $H-Ras^{V12}$ protein induces DNA synthesis and ethanol treatment did not interfere with the DNA synthesis. The antiproliferative toxicity of ethanol was rescued by antioxidants, such as N-acetylcysteine and 4-methlpyrazole. These results indicate that the antiproliferative action site of ethanol toxicity lies upstream or is independent of Ras and ethanol exerts its toxicity through a free radical formation.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.28 no.1
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• pp.245-259
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• 1998

Cellular transforming genes have been identified in a number of different tumor cell lines and tumor types. A significant number of these oncogenes belong to the ras gene family. The ras gene family consists of three closely related genes:H-ras, K-ras and N-ras which code for a related 21 kDa protein. Mutations in codon 12, 13 and 61 of one of the three ras genes convert these genes into acute oncogenes. The presence of H-ras gene mutations has important prognostic implications in various tumors. Each genomic DNA was isolated from tumors induced by implantation with DMBA, or by treatment with DMBA -implantation/irradiation. When genome DNA was transfected into NIH 3T3 cells and investigated by two-step PCR-RFLP, the fOllowing results were concluded: 1. Transformation foci developed in two groups when the genome DNA of two experimental groups were transfected into NIH 3T3 cells. 2. Transformation efficiency was 0.01-0.02 foci/㎍DNA in the experimental group with the DMBA-implantation, 0.01-0.03 foci/㎍lgDNA in the experimental group with the DMBA-implantation/irradiation according to results of transfection assay. 3. When the point mutation of H-ras gene was investigated by a two-step PCR-RFLP, there was 13.9％ (5/36) in the experimental group with the DMBA implantation, 15.4 ％ (6/39) in the experimental group with the DMBA -implantation/irradiation. 4. The point mutation in codon 12 and 61 of H-ras was 5.6％(2/36) and 8.3％(3/36) in the experimental group with the DMBA implantation. 5. The point mutation in codon 12 and 61 of H-ras gene was 7.7％(3/39) in the experimental group with the DMBA -implantation/irradiation.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.223.1-223.1
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• 2003

Many studies have identified the phosphatidylinositol 3-kinase (PI3K) as a key regulator for various cellular functions including cell survival, growth and motility. We have previously shown that H-ras, but not N-ras. induces invasiveness and motility in human breast epithelial cells (MCF10A), while both H-ras and N-ras induce transformed phenotype. In the present study, we wished to investigate the functional role of PI3K pathway in H-ra-induced invasive phenotype and motility of MCF10A cells. (omitted)

• Archives of Pharmacal Research
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• v.24 no.4
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• pp.349-354
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• 2001

Curcumin, a dietary pigment in turmeric, posseses anti-carcinogenic and anti-metastatic properties. The present study was conducted to study in vitro chemopreventive effects of curcumin in transformed breast cells. Here, we show that curcumin inhibits H-ras-induced invasive phenotype in MCF10A human breast epithelial cells (H-ras MCF10A) and downregulates matrix metalloproteinase (MMP)-2 dose-dependently. Curcumin exerted cytotoxic effect on H-ras MCF10A cells in a concentration-dependent manner. Curcumin-induced cell death was mainly due to apoptosis in which a prominent downregulation of Bcl-2 and upregulation of Bax were involved. We also suggest a possible involvement of caspase-3 in curcumin-induced apoptosis. Curcumin treatment resulted in the production of reactive oxygen species (ROS) in H-ras MCF10A cells. Apoptotic event by curcumin was significantly inhibited by pretreatment of an antioxidant N-acetyl-$_L$-cysteine (NAC), suggesting redox signaling as a mechanism responsible for curcumin-induced apoptosis in H-ras MCF10A cells. Taken together, our results demonstrate that curcumin inhibits invasion and induces apoptosis, proving the chemopreventive potential of curcumin .

• Toxicological Research
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• v.14 no.4
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• pp.569-575
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• 1998

One of the most frequent dejects in human cancer is the uncontrolled activation of the ms-signaling pathways. Significant evidence has accumulated to directly implicate members of the matrix metalloproteinases (MMPs) in tumor invasion and metastasis formation. We have previously shown that MMP-9 expression was significantly enhanced in the ras-tranfected HT1080 human fibrosarcoma cells at the mRNA level. In the present study, we investigated the roles of MMP-2 and -9 on the H-ras-induced invasive phenotypes of MCF 10A human breast epithelial cells and HT 1080 human fibrosarcoma cells. We show that H-ras is able to induce or enhance a signaling pathway leading to the enhancement of an invasive phenotype in both MCF10A and HT1080 cells as determined by matrigel invasion assay. We then examined the effect of H-ras activation on the expression of MMP-2 and -9 by measuring enzymatic activities and mRNA levels. Our data clearly demonstrated that H-ras prominently induces expression of MMP-2 in MCF10A cells, while it efficiently up regulates MMP-9 in HT1080 cells. Taken together, these findings suggest that the correlation between ras-mediated invasiveness and enhanced expression of MMPs may be cell type-specific: MMP-9 is closely associated with the invasive phenotype induced by ras activation in fibrosarcoma cells, whereas MMP-2 is more likely associated with it in epithelial cells.