• The Plant Pathology Journal
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• 제27권1호
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• pp.20-25
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• 2011

Gibberella zeae (anamorph: Fusarium graminearum), a homothallic (self-ferile) ascomycete with ubiquitous geographic distribution, causes serious diseases in several cereal crops. Ascospores (sexual spores) produced by this fungal pathogen have been suggested as the main source of primary inoculum in disease development. Here, we report the function of a gene designated GzRUM1, which is essential for ascospore formation in G. zeae. The deduced product of GzRUM1 showed significant similarities to the human retinoblastoma (tumor suppressor) binding protein 2 and a transcriptional repressor, Rum1 in the corn smut fungus (Ustilago maydis). The transcript of GzRUM1 was detected during the both vegetative and sexual stages, but was more highly accumulated during the latter stage. In addition, no GzRUM1 transcript was detected in a G. zeae strain lacking a mating-type gene (MAT1-2), a master regulator for sexual development in G. zeae. Targeted deletion of GzRUM1 caused no dramatic changes in several traits except ascospore formation. The ${\Delta}$GzRUM1 strain produced perithecia (sexual fruit bodies) but not asci nor ascospores within them. This specific defect leading to an arrest in ascospore development suggests that GzRUM1, as Rum1 in U. maydis, functions as a transcriptional regulator during sexual reproduction in G. zeae.

• Animal cells and systems
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• 제6권3호
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• pp.271-275
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• 2002

Rph1 and Gisl are damage-responsive repressors involved in PHR1 expression. They have two $C_{2}$H/ sub 2/ zinc finger motifs as putative DNA binding domains and N-terminal conserved domain with unknown function. They are also found in the human retinoblastoma binding protein 2 and the mouse jumonji- encoded protein. The repressors are able to bind to A $G_{4}$ sequence within a 39-bp sequence called upstream repressing sequence of PHR1 promoter (UR $S_{PHR1}$) responsible for the damage-response of PHR1. We report here that Rph1 is predominantly localized in the nucleus as examined by fluorescence microscopic analysis with GFP-Rph1 fusion protein. On the basis of the fact that the A $G_{4}$ sequence that is recognized by Rph1 and Gisl is also recognized by Msn2 and Msn4 in a process of stress response, we a1so tried to examine the in vivo function of A $G_{4}$ and the role of Msn2 and Msn4 in PHR1 expression. Our results demonstrate that Msn2 and Msn4 are actually required for the basal transcription of PHR1 expression but not for its damage induction. When A $G_{4}$ sequence was inserted into the minimal promoter of the cyc1-LacZ reporter, the increased LacZ expression was observed indicating its involvement in transcriptional activation. The data suggest that the A $G_{4}$ is primarily required for basal transcriptional activation of PHR1 or CYC1 promoter through the possible involvement of Msn2 and Msn4. However, since the A $G_{4}$ is also involved in the repression of PHR1 via Rphl and Gisl, it is proposed that A $G_{4}$ functions as either URS or upstream activating sequence (UAS) depending on the promoter context.t.

• BMB Reports
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• 제34권1호
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• pp.80-84
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• 2001

The human papillomavirus E7 protein can form a specific complex with a retinoblastoma tumor suppressor gene product (p105-Rb) that results in the release of the E2F transcription factor, which is critical for the growth-deregulation and transforming properties of the viral E7 oncoprotein. In an attempt to apply interaction between the E7 oncoprotein and a target cellular protein Rb for an in vitro screening system for drugs against human papillomavirus infection, we primarily investigated the E7Rb binding through a pull down assay and enzyme-linked immunosorbent assay. The pull down assay showed that both glutathione S-transferase-tagged E7 and His-tagged E7 immobilized on resins specifically produced complexes with bacterially expressed Rb in a dose-dependent manner, as determined by immunoblot analyses. This result coincided with that of an enzyme-linked immunosorbent assay, which is a useful system for the mass screening of potential drugs. Taken together, this screening system (based on the interaction between E7 and Rb) can be a promising system in the development of drugs against cervical cancers caused by human papillomavirus infection.

• BMB Reports
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• 제40권5호
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• pp.765-772
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• 2007

Eosinophils act as effectors in the inflammatory reactions of allergic diseases including atopic dermatitis. Atopic dermatitis patients and others with allergic disorders suffer from eosinophilia, an accumulation of eosinophils due to increased survival or decreased apoptosis of eosinophils. In this study, a differential phosphoproteome analysis of AML14.3D10 eosinophil cell line after treatment with IL-5 or dexamethasone was conducted in an effort to identify the phosphoproteins involved in the proliferation or apoptosis of eosinophils. Proteins were separated by 2-DE and alterations in phosphoproteins were then detected by Pro-Q Diamond staining. The significant quantitative changes were shown in nineteen phosphoproteins including retinoblastoma binding protein 7, MTHSP75, and lymphocyte cytosolic protein 1. In addition, seven phosphoproteins including galactokinase I, and proapolipoprotein, were appeared after treatment with IL-5 or dexamethasone. Especially, the phospho-APOE protein was down-regulated in IL-5 treated AML14.3D10, while the more heavily phosphorylated APOE form was induced after dexamethasone treatment. These phosphoproteome data for the AML14.3D10 cell line may provide clues to understand the mechanism of eosinophilia as well as allergic disorders including atopic dermatitis.

• IMMUNE NETWORK
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• 제2권1호
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• pp.60-64
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• 2002

Background: Chemotherapy with 5-fluorouracil (5-FU) has been one of the mainstay in breast cancer treatment. The effects of high dose 5-FU on cell cycle regulation were studied in breast caner cells. Methods: A breast cancer cell line MCF-7 was used. Protein expressions of G1/S cyclins, $p21^{Waf1/Cip1}$, cdk2, E2F1 and retinoblastoma were tested by western blot analysis. Immunoprecipitation and immune complex kinase assay were done for the assessment of E2F1/RB interacton and the activity of cdk2 respectively. Results: $p21^{Waf1/Cip1}$ expression was barely detectable in control cells. With addition of 5-FU level of $p21^{Waf1/Cip1}$ were induced and cyclin D3 level was decreased as cell growth decreases. In accordance with increased expression of $p21^{Waf1/Cip1}$, cyclin E-associated cdk2 kinase activity was reduced. Retinoblastoma protein (RB) became dephosphorylated and E2F-1 binding activity with RB was increased. Conclusion: In this situation of high concentration of 5-FU breast cancer cells tend to be G1/S cell cycle arrested. Overexpression of $p21^{Waf1/Cip1}$ and dephosphorylation of RB may mediate the effectss of 5-FU by inhibiting E2F-1 activity, which contributes to G1/S cell cycle arrest. These results could be an indicating landmark for further study of high dose chemotherapy with 5-FU.

• 생명과학회지
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• 제19권7호
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• pp.871-877
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• 2009

대표적인 histone deacetylase inhibitor 저해제의 일종일 sodium butyrate에 의한 인체백혈병 U937세포의 증식 억제에 관한 기전 연구를 세포주기 조절 측면에서 조사하였다. MTT assay 및 flow cytometry 분석을 통하여 sodium butyrate의 처리 농도 증가에 따른 U937 세포의 증식억제는 세포주기 G1 arrest 및 apoptosis 유발에 의한 것임을 확인하였다. RT-PCR및 Western blotting 결과에서 sodium butrate에 의한 G1 arrest는 세포주기 G1기에서 S기로의 진입에 중요한 역할을 하는 cyclin D1, E, A, cyclin-dependent kinase (Cdk) 4 및 Cdk6발현의 저해와 p21 및 p27과 같은 Cdk inhibitor의 발현 증가와 연관성이 있었다. Sodium butyrate는 또한 retinoblastoma protein (pRB)및 p130 단백질의 인산화를 저해시켰으나, S기 진행에 중요한 전사조절인자인 E2F-1 및 E2F-4의 의 발현에는 큰 영향이 없었다. 그러나 sodium butyrate에 의한 pRB 및 p130단백질의 인산화 저해는 pRB와 E2F-1및 p130과 E2F-4와의 결합력을 증사시켰다. 본 연구의 결과는 U937세포의 증식억제에 pRB/p130 인산화 억제 및 Cdk inhibitors의 발현 증가가 중요한 역할을 하고있음을 보여주는 것으로, sodium butyrate의 항암기전 이해에 중요한 자료가 될 것이다.

• BMB Reports
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• 제36권2호
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• pp.223-229
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• 2003

The product of a tree (Tabebuia avellanedae) from South America, $\beta$-lapachone, is known to exhibit various pharmacological properties, the mechanisms of which are poorly understood. The aim of the present study was to further elucidate the possible mechanisms by which $\beta$-lapachone exerts its anti-proliferative action in cultured human prostate cancer cells. We observed that the proliferation-inhibitory effect of $\beta$-lapachone was due to the induction of apoptosis, which was confirmed by observing the morphological changes and cleavage of the poly(ADP-ribose) polymerase protein. A DNA flow cytometric analysis also revealed that $\beta$-lapachone arrested the cell cycle progression at the G1 phase. The effects were associated with the down-regulation of the phosphorylation of the retinoblastoma protein (pRB) as well as the enhanced binding of pRB and the transcription factor E2F-1. Also, $\beta$-lapachone suppressed the cyclindependent kinases (Cdks) and cyclin E-associated kinase activity without changing their expressions. Furthermore, this compound induced the levels of the Cdk inhibitor $p21^{WAF1/CIP1}$ expression in a p53-independent manner, and the p21 proteins that were induced by $\beta$-lapachone were associated with Cdk2. $\beta$-lapachone also activated the reporter construct of a p21 promoter. Overall, our results demonstrate a combined mechanism that involves the inhibition of pRB phosphorylation and induction of p21 as targets for $\beta$-lapachone. This may explain some of its anticancer effects.

• 한국해양바이오학회지
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• 제1권4호
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• pp.243-251
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• 2006

Dideoxypetrosynol A, a polyacetylene from the marine sponge Petrosia sp., is known to exhibit significant selective cytotoxic activity against a small panel of human tumor cell lines, however, the mechanisms of which are poorly understood. In the present study, it was investigated the further possible mechanisms by which dideoxytetrosynol A exerts its anti-proliferative action in cultured human leukemia cell line U937. We observed that the proliferation-inhibitory effect of dideoxypetrosynol A was due to the induction of G1 arrest of the cell cycle and apoptosis, which effects were associated with up-regulation of cyclin D1 and down-regulation of cyclin E without any change in cyclin-dependent-kinases (Cdks) expression. Dideoxypetrosynol A markedly induced the levels of Cdk inhibitor p16/INK4a expression. Furthermore, down-regulation of phosphorylation of retinoblastoma protein (pRB) by this compound was associated with enhanced binding of pRB and the transcription factor E2F-1. The increase in apoptosis was associated with a dose-dependent up-regulation in pro-apoptotic Bax expression and activation of caspase-3 and caspase-9. Dideoxytetrosynol A decreased the levels of cyclooxygenase (COX)-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with a decrease in prostaglandin E2 (PGE2) synthesis. Furthermore, dideoxytetrosynol A treatment markedly inhibited the activity of telomerase, and the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by dideoxytetrosynol A treatment in a dose-dependent fashion. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of dideoxytetrosynol A.

• 생명과학회지
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• 제13권5호
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• pp.642-650
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• 2003

인체 신경아세포종의 성장에 미치는 cAMP의 영향을 조사하기 위하여 Ewing's sarcoma 세포주인 CHP-100 세포에 dibutyry1-cAMP 및 8-bromo-cAMP를 처리하였다. 두 종류의 cAMP analog처리 시간 증가에 따라 CHP-100 세포의 증식이 처리 시간 의존적으로 억제되었으며, 이는 핵의 형태변화 및 DNA 단편화 현상을 수반한 apoptosis 유발과 연관성이 있었다. 또한 DNA flow cytometry 분석결과 cAMP는 세포주기 G1기 특이적 arrest를 유발하였다. cAMP 처리에 의하여 retinoblastoma 단백질(pRB)의 인산화가 억제되었으며, 전사조절인자 E2F-1과의 결합이 증대되었다. cAMP는 cyclin-dependent kinase (Cdk) 2 및 cyclin E 단백질의 발현변화에는 영향을 미치지 않았으나, 그들의 kinase 활성은 처리시간 의존적으로 매우 감소되었다. 또한 cAMP 처리에 의하여 Cdk inhibitor인 $p21^{WAF1/CIP1$의 발현이 증가되었으며, 증가된 p21 단백질은 Cdk2와 강한 결합을 형성하고 있었다. 이상의 결과에서 cAMP의 암세포 성장억제 효과에 pRB 및 p21이 매우 중요한 역할을 함을 알 수 있었다.

• 동의생리병리학회지
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• 제22권4호
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• pp.821-828
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• 2008

Onchungeum, a herbal formula, which has been used for treatment of anemia due to bleeding, discharging blood and skin disease. In the present study, it was examined the effects of extract of Onchungeum (OCE) on the growth of human hepatocarcinoma cell lines Hep3B (p53 null type) and HepG2 (p53 wild type) in order to investigate the anti-proliferative mechanism by OCE. Treatment of Hep3B and HepG2 cells to OCE resulted in the growth inhibition in a dose-dependent manner, however Hep3B cell line exhibited a relatively strong anti-proliferative activity to OEC. Flow cytometric analysis revealed that OCE treatment in Hep3B cells caused G1 phase arrest of the cell cycle, which was associated with various morphological changes in a dose-dependent fashion. RT-PCR and immunoblotting data revealed that treatment of OCE caused the down-regulation of cyclin D1 expression, however the levels of cyclin E expression were not changed by OCE. The G1 arrest of the cell cycle was also associated with the induction of Cdk inhibitor p27 by OCE. Because the p53 gene is null in Hep3B cells, it is most likely that the induction of p21 is mediated through a p53-independent pathway. Moreover, p27 detected in anti-Cdk4 and anti-Cdk2 immunoprecipitates from the OCE-treated cells, suggesting that OCE-induced p27 protein blocks Cdk kinase activities by directing binding to the cyclin/Cdk complexes. Furthermore, OCE treatment potently suppresses the phosphorylation of retinoblastoma proteins and the levels of the transcription factor E2F-1 expression. Taken together, these results indicated that the growth inhibitory effect of OCE in Hep3B hepatoma cells was associated with the induction of G1 arrest of the cell cycle through regulation of several major growth regulatory gene products.