Evidence for the Ras-Independent Signaling Pathway Regulating Insulin-Induced DNA Synthesis

Jhun, Byung H. The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.2

The existence of the Ras-independent signal transduction pathway of insulin leading to DNA synthesis was investigated in Rat-1 fibroblasts overexpressing human insulin receptor (HIRc-B) using the single-cell microinjection technique. Microinjection of a dominant-negative mutant Ras protein into quiescent HIRc-B cells inhibited the DNA synthesis stimulated by insulin. Microinjection of oncogenic H-Ras protein (H-Ras) (0.1㎎/ml) induced DNA synthesis by 35%, whereas that of control-injected IgG was induced by 20%. When the maginal amount of oncogenic H-Ras protein was coinjected with a dominant-negative mutant of the H-Ras protein(H-Ras), DNA synthesis was 35% and 74% in the absence and presence of insulin, respectively. This full recovery of DNA synthesis by insulin suggests the existence of the Ras-independent pathway. The same recovery was observed in the cells coinjected with either H-Ras H-Ras plus SH2 domain of the p85 subunit of PI3-kinase(p85) or H-Ras plus H-Ras plus interfering anti-Shc antibody. When coinjected with a dominant-negative Rac1, the DNA synthesis induced by the Ras-independent pathway was blocked. These results indicate that the Ras-independent pathway of insulin leading to DNA synthesis exists, bypassing the p85 of PI3-kinse and Shc protein, and requires Rac1 protein.

Evidence for the Ras-Independent Signaling Pathway Regulating Insulin-Induced DNA Synthesis

Jhun, Byung H . 생화학분자생물학회 2000 BMB Reports Vol.32 No.2

The existence of the Ras-independent signal transduction pathway of insulin leading to DNA synthesis was investigated in Rat-1 fibroblasts overexpressing human insulin receptor (HIRc-B) using the single-cell microinjection technique. Microinjection of a dominant-negative mutant Ras^(N17) protein into quiescent HIRc-B cells inhibited the DNA synthesis stimulated by insulin. Microinjection of oncogenic HRas^(V12) protein (H-Ras^(V12)) (0.1 ㎎/㎖) induced DNA synthesis by 35%, whereas that of control-injected IgG was induced by 20%. When the marginal amount of oncogenic H-Ras^(V12) protein was coinjected with a dominant-negative mutant of the H-Ras^(V12) protein (H-Ras^(N17)), DNA synthesis was 35% and 74% in the absence and presence of insulin, respectively. This full recovery of DNA synthesis by insulin suggests the existence of the Ras-independent pathway. The same recovery was observed in the cells coinjected with either H-Ras^(V12) plus H-Ras^(V12) plus SH2 domain of the p85 subunit of PI3-kinase (p85^(SH2-N)) or H-Ras^(V12) plus H-Ras^(N17) plus interfering anti-Shc antibody. When coinjected with a dominant-negative Rac1^(N17), the DNA synthesis induced by the Ras-independent pathway was blocked. These results indicate that the Rasindependent pathway of insulin leading to DNA synthesis exists, bypassing the p85 of PI3-kinase and Shc protein, and requires Rac1 protein.

Evidence for the Ras-Independent Signaling Pathway Regulating Insulin-Induced DNA Synthesis

Jhun, Byung-H. Korean Society for Biochemistry and Molecular Biol 1999 Journal of biochemistry and molecular biology Vol.32 No.2

The existence of the Ras-independent signal transduction pathway of insulin leading to DNA synthesis was investigated in Rat-1 fibroblasts overexpressing human insulin receptor (HIRc-B) using the single-cell microinjection technique. Microinjection of a dominant-negative mutant $Ras^{N17}$ protein into quiescent HIRc-B cells inhibited the DNA synthesis stimulated by insulin. Microinjection of oncogenic H-$Ras^{V12}$ protein ($H-Ras^{V12}$) (0.1 mg/ml) induced DNA synthesis by 35%, whereas that of control-injected IgG was induced by 20%. When the marginal amount of oncogenic H-$Ras^{V12}$ protein was coinjected with a dominant-negative mutant of the H-Ras protein ($Ras^{N17}$), DNA synthesis was 35% and 74% in the absence and presence of insulin, respectively. This full recovery of DNA synthesis by insulin suggests the existence of the Ras-independent pathway. The same recovery was observed in the cells coinjected with either H-$Ras^{V12}$ plus H-$Ras^{N17}$ plus SH2 domain of the p85 subunit of PI3-kinase ($p85^{SH2-N}$) or H-$Ras^{V12}$ plus H-$Ras^{N17}$ plus interfering anti-Shc antibody. When co-injected with a dominant-negative H-$Ras^{N17}$, the DNA synthesis induced by the Ras-independent pathway was blocked. These results indicate that the Ras-independent pathway of insulin leading to DNA synthesis exists, bypassing the p85 of PI3-kinase and Shc protein, and requires Rac1 protein.

Farnesyl transferase 억제제인 YH3938 및 YH3945에 의한 Ras 발암원성 억제

Myung-Ju Oh(오명주),Nong Yeon Kim(김농연),Su-Eun Lim(임수은),Young-Hwa Chung(정영화),Byung H Jhun(전병학) 한국생명과학회 2010 생명과학회지 Vol.20 No.2

Ras 유전자는 30%의 인간암에서 변이가 발견되며 세 종류의 isoform, H-Ras, K-Ras 및 N-Ras로 구성되어 있다. Ras 단백질의 CAAX motif에 farnesylation과 같은 번역 후 변형은 Ras의 활성에 필수 요소이다. 본 연구에서는 새로운 farnesyl transferase 억제제인 YH3938과 YH3945의 발암원성 H-Ras, K-Ras 및 N-Ras의 작용에 대한 영향을 조사하였다. YH3938과 YH3945는 발암원성 H-Ras에 의해 형질전환된 Rat2 세포의 증식과 형태 변화를 억제하였으나 K-Ras에 대해서는 효과가 없었다. N-Ras에 대해서는 약한 영향이 있었다. H-Ras와 N-Ras에 의한 SRE promoter 활성화는 YH3938과 YH3945에 의해 억제되었으나, K-Ras에는 영향이 없었다. Ras 단백질의 bandshift 분석을 통해 YH3938은 H-Ras와 N-Ras의 번역 후 변환을 억제하였으나, K-Ras에는 영향이 없었다. YH3945는 H-Ras의 변환에만 영향이 있었다. 결론적으로 YH3938과 YH3945는 H-Ras의 farnesylation을 억제하여 그 발암원성을 억제하며, YH3938은 N-Ras 작용을 농도의존적으로 억제하며, K-ras에 대해서는 영향이 없음을 알 수 있었다. Ras genes are responsible for up to 30% of human tumor mutations and are composed of three isoforms: H-Ras, K-Ras and N-Ras. The post-translational modification of the CAAX motif of the Ras protein is essential in Ras actions. In the present study, we studied the effects of novel farnesyl transferase inhibitors (FTIs), YH3938 and YH3945, on the actions of oncogenic mutants of H-Ras, K-Ras and N-Ras. YH3938 and YH3945 completely reverted the proliferation and morphology of oncogenic H-Ras-transformed Rat2 cells, but not of oncogenic K-Ras-transformed Rat2 cells. Oncogenic N-Rastransformed Rat2 cells were slightly affected. Activation of SRE promoters by oncogenic H-Ras and N-Ras, but not by K-Ras, were inhibited by treatment with YH3938 and YH3945. Using bandshift analysis, YH3938 suppressed the processing of oncogenic H-Ras and N-Ras, but not that of oncogenic K-Ras protein. YH3945 only inhibited the processing of H-Ras. From these results, we conclude that YH3938 and YH3945 specifically inhibit actions of oncogenic H-Ras through inhibition of its farnesylation, that YH3938 also inhibits N-Ras activity in a dose-dependent manner, and that these drugs have no effect on oncogenic K-Ras activity.

Ginsenoside Rb1에 의한 발암유전자 ras의 활성 억제

이선주,전병학 대한암예방학회 2003 Journal of cancer prevention Vol.8 No.2

Chronic Treatment of Kthanol Inhibits Proliferation of Normal Fibroblasts,but Not Oncogenie ras-Transformed Cells

Gu, Young Hwa,Park, Mi Sun,Jhun, Byung H . 한국응용약물학회 1998 Biomolecules & Therapeutics(구 응용약물학회지) Vol.6 No.4

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.

Kimchi and an Active Component, β-Sitosterol, Reduce Oncogenic H-Ras(V12)-Induced DNA Synthesis

( Kun Young Park ),( Eun Ju Cho ),( Sook Hee Rhee ),( Keun Ok Jung ),( Sun Ju Yi ),( Byung H. Jhun ) 부산대학교 김치연구소 2003 김치의 과학과 기술 Vol.9 No.-

Kimchi and an Active Component, β-Sitosterol, Reduce Oncogenic H-Rasv12-Induced DNA Synthesis

Kun-Young Park,Eun-Ju Cho,Sook-Hee Rhee,Keun-Ok Jung,Sun-Ju Yi,Byung H. Jhun 한국식품영양과학회 2003 Journal of medicinal food Vol.6 No.3

The Korean fermented vegetable food, kimchi, has been demonstrated to have anticancer functional proper-ties. This study examined the effect of kimchi samples, methanol extracts of commercially grown baechu cabbage kimchi(CK) and organically grown baechu cabbage kimchi (OK), as well as the dichloromethane fraction (DCM fr.) from CK, andthe active compound (AC), which has ben identified as largely b-sitosterol, from DCM fr., on the Ras-dependent signalingpathway. CK, OK, and DCM fr. exhibited a greater inhibition against the proliferation of Rat2 fibroblasts transformed withRasv12 (HO6) than parental Rat2 fibroblasts. In addition, OK and DCM fr. showed a higher inhibitory effect than CK. Fur-thermore, we employed the single-cell microinjection technique, combined with 3 bromo-5’ -deoxyuridine incorporation, toexamine the effects of kimchi samples on DNA synthesis induced by microinjected oncogenic Ras v12. When the DCM fr. andAC were used to treat Rat1 fibroblasts overexpressing human insulin receptors (HIRc-B) and microinjected with oncogenicH-Rasv12, the DNA synthesis of injected cells was decreased, sugesting that kimchi might block the signaling pathway ofoncogenic Ras v12, thus preventing the proliferation of transformed cells. This study provides additional evidence that kimchiand its active components, including b-sitosterol, have potential in both the prevention and treatment of cancer, and presentsconvincing evidence that the anticancer effects may be a result of an inhibition of Ras oncogene signaling.

Anti-Proliferative Effect of Ethanol on Normal and Cancer Cells

Myung-Ju Oh(오명주),Jihyun Kim(김지현),Su-Hyun Park(박수현),Young-Hwa Jeong(정영화),Kun Wang(왕쿤),Byung-Wook Cho(조병욱),Byung H. Jhun(전병학) 한국생명과학회 2012 생명과학회지 Vol.22 No.4

에탄올은 사람에 대한 발암물질로 잘 알려져 있다. 또한 여러 조직이나 세포에서의 에탄올에 의한 세포증식억제효과도 잘 알려져 있다. 본 연구에서는 여러 암세포에서 에탄올에 의한 세포증식억제 효과를 조사하였는데 특히 발암원성 ras로 형질전환되거나 미세주입된 세포에서의 영향을 조사하였다. 에탄올은 여러 정상세포들의 증식을 억제하였다. 반면에 여러 암세포나 발암원성 Ras에 의한 세포증식은 억제하지 못 하였다. 또한 발암원성단백질의 세포내 미세주사에 의한 DNA합성 유도도 에탄올에 의해 억제 되지 않았다. 이러한 에탄올의 세포증식 억제 효과는 N-acetylcysteine이나 4-methylpyrazole과 같은 항산화제에 의해 제거되었다. 이러한 실험 결과는 에탄올에 의한 세포증식억제 효과는 Ras단백질의 upstream에 있거나 또는 Ras와 독립적으로 작용하며, 활성산소 형성과 밀접한 관계가 있다는 것을 알려준다. Ethanol is known as being carcinogenic to humans. In addition, the anti-proliferative effects of ethanol have been described for a variety of tissues and cells. In this study, we investigated the anti-proliferative effects of ethanol on various cancer cells, particularly on oncogenic ras-transformed or -injected cells. Ethanol treatment inhibited the cell proliferation of normal control cells, but did not suppress the proliferation of various cancer cells and oncogenic ras-transformed cells. Furthermore, ethanol treatment did not interfere with DNA synthesis, which was induced by microinjecting the oncogenic H-RasV12 protein. The anti-proliferative effect of ethanol was rescued by antioxidants, such as N-acetylcysteine and 4-methlpyrazole. These results suggest that ethanol cytotoxicity is exerted through free radical formation, and that the anti-proliferative action site of ethanol cytotoxicity either lies upstream, or is independent of Ras.

Phenylarsine Oxide Causes an Insulin-dependent, GLUT4-specific Degradation in Rat Adipocytes

Jhun, Byung H.,Hah, Jong S.,Jung, Chan Y. 이화여자대학교 생명과학연구소 1991 생명과학연구논문집 Vol.2 No.-

An incubation of rat adipocytes with phenylarsine oxide(PAO) and then with insulin caused an inhibition of 3-0-methylglucose equilibrium exchange flux and a parallel reduction in cellular GLUT4 content detected by Western blots. Both the transport inhibition and the GLUT4 reduction were saturable with an increasing concentration of PAO showing essentially an identical K. value of 35 ㎛. Both effects were not observed in the absence of insulin or if cells were incubated with insulin first. The reduction was specific to GLUT4; the immunoreactivities of GLUT1, insulin receptor, and clathrin were not affected in these experiments. The GLUT4 reduction occurred only in intact cells and was not observed in homogenized cells or fractionated membranes. GLUT4 reduction was not observed in the presence of chloroquine or at 18℃suggesting involvement of the lysosomal pathway. Based on these results, we propose that there is a PAO-sensitive protein mechanism that controls an insulin-dependent GLUT4 degradation pathway in adipocytes. This protein mechanism and the GLUT4 degradation pathway may play an important role in determining the steady-state GLUT4 level in the insulin-sensitive peripheral tissues in normal and diseased states.