MOLECULAR MECHANISM THAT RETINOIC ACID RECEPTOR REGULATES THE TRANSCRIPTIONAL ACTIVITY OF C - JUN

엄수종 ( Soo Jong Um,Harbers Mattias,Pierre Chambon ) 생화학분자생물학회 1997 생화학분자생물학회 춘계학술발표논문집 Vol.- No.-

자궁경부암 세포에서 retinoid / interferon 에 의한 성장 억제 조절의 새로운 분자생물학적 기전

김수평(Soo Pyung Kim),남궁성은(Sung Eun Namkoong),김승조(Seung Jo Kim),김은주(Eun Joo Kim),엄수종(Soo Jong Um),박종섭(Jong Sup Park),이근호(Keun Ho Lee),김찬주(Chan Joo Kim) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.3

N/A Objective: Retinoic acids (RAs) and interferons (IFNs) have been implicated in the growth regulation of cervical cancer cells, which was suggested by clinical trials and in vitro experiments. However, the molecular mechanisms of growth regulation are not fully defined, The purpose of this study is to assess the effect of RA and/or IFN on human cervical carcinoma cells in vitro and to analyze their action mechanisms in HPV-positive cervical carcinoma cells by molecular biologic studies. Methods: HPV-positive (CaSki, HeLa), HPV-negative (C33A, HT-3), and non-cervical cancer Cos-1 cell lines were treated with RA and/ar IFN. Their effects on cell growth were evaluated by the cell pmliferation assay and the following BrdU DNA incorporation assay. The molecular mechanism was further investigated by a series of immunoblottings and transient cotransfection assays, which were conducted in HeLa cells and C33A cells using the CAT reporter gene assay. To observe the down regulation of HPV E6/E7 gene expression by RA/IFN, reverse transcription-polymerase chain reaction (RT-PCR) was perforned. Results: The powth of RA-treated cells was less suppressed than that of IFN-treated cells. Combined treatment of RA and IFN leads to additive effect on the growth suppression of HeLa and CaSki cells. The proliferation activity was most severely reduced in Hela cells by treatment of both all-trans-RA (AtRA) and IFN-r. Combined treatment of AtRA/IFN-r causes a great increase in the level of interferon regulatory factor-1 (IRF-1) protein in HeLa cells, whereas no induction of IRF-1 was observed in C33A cells. The CAT gene expression for IRF-1 was greatly induced by IFN-r in HeLa cells. Immunoblotting assays shows the concurrent induction of p21 CDK inhibitor and dephosphorylation of Rb protein in HeLa cells. In RT-PCR, an individual treatment of either RA or IFN reduced HPV E6/E7 mRNA levels and significantly cooperative when both RA and IFN were treated. By deaeasing E6 levels, the p53 level was increased in HeLs cells treated with RA and/or IFN. Transient cotransfection of IRF-1 and p53 as the transcription factors leads to the cooperative activation of a common p21 promoter to regulate the cell cycle. Conclusion: RA/IFN suppressed the growth of HPV-positive cervical cancer cells. When they were both #treated, additive suppressive effects were observed in cellular proliferation as well as DNA synthesis. The growth suppressive effect is likely to be related to the increased expression of IRF-1 and p21 (antitumoral effect; p53-independent). The down regulation of HPV E6 gene suppression may account for the resultant increase of p53 levels (antiviral effect; p53-dependent). Both induced IRF-1 and p53 cooperatively augument tbe suppession of p21 CDK inhibitor, which results in dephosphorylation of pRb. Although clinical effects are likely complex and may include interactions of in vitro growth inhibitory effects with immunomodulatory and antiangiogeaetic effect, tbese results suggest the optimal clinical role for the combination of RA/IFN in the treatment of cervical canccers.

자궁경부암을 유발하는 HPV E6 단백질에 의한 p73 단백질의 불활성화 : p53과 무관한 E6의 새로운 기능

남궁성은(Sung Eun Namkoong),김승조(Seung Jo Kim),김은주(Eun Joo Kim),엄수종(Soo Jong Um),박종섭(Jong Sup Park) 대한산부인과학회 1998 Obstetrics & Gynecology Science Vol.41 No.11

N/A Objective: Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 and E7 oncoproteins play major roles by inactivation of cellular p53 and pRb tumor suppressor proteins, respectively. However, it has been recently suggested that p53 and/or pRb-independent functions of E6 and E7 are involved in cervical carcinogenesis. The purpose of this study is to identify novel a cellular target, p73, of E6 and to determine how E6 inactivates p73 function, Methods: The interaction between E6 and p73 were identified by the yeast two-hybrid assay in vivo and the GST pull-down assay in vitro. The function of the interaction was determined by transient transfections using p21 promoter-CAT reporter plasmid. The molecular mechanism underlying the functional significance of the interaction was further assessed by in vivo and in vitro protein degradation assays, and gel mobility shift assays. Results: Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. Transactivation domain (amino acid residues 1-49) is found to be absolutely required for this interaction. Transient co-expression of E6 significantly inhibits the p73-mediated activation of p21 promoter in a p53-defective C33A cell line. Using Ga14-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. The protein degradation assays in vivo and in vitro indicate that p73, unlike p53, is not susceptible to E6-dependent proteolysis. Conclusion: Throughout this study, we identified p73 as a novel cellular target of HPV-E6 protein and found that E6 binds p73 through the amino-terminal transactivation domain, and inhibits its transactivation function independent of the protein degradation and DNA binding. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated cellular transformation.

cDNA microarray를 이용한 HPV-16 E6와 연관된 유전자의 발현 분석

명진 ( Jin Myeong ),라선영 ( Sun Young Rha ),이명진 ( Myoung Jin Lee ),엄수종 ( Soo Jong Um ),남궁성은 ( Sung Eun Namkoong ),박종섭 ( Jong Sup Park ) 대한산부인과학회 2002 Obstetrics & Gynecology Science Vol.45 No.12

목적 : 이 연구는 자궁경부암의 주요 발암 원인 인자로 알려진 HPV-16 E6의 발현이 세포내 유전자들의 전사조절에 미치는 영향을 관찰하기 위해서 시행되었다. 연구 방법 : HPV-16 E6를 안정적으로 발현하는 A549E6와 RC10.1 세포주를 실험군으로 사용하여 cDNA microarray 실험을 시행하였다. 결과 : A549E6 세포주에서 HPV-16 E6의 세포내 발현으로 증가된 발현양상을 보인 주요 유전자들로는 GTPase-activatin Objective : To examine the effect of HPV-16 E6 expression on the transcription of cellular genes, we used cDNA microarray in HPV-16 E6 transfected stable cancer cell lines. Methods : Using cDNA microarray consisting of 1,024 genes, we have performed a sys

암과 비타민 A

엄수종 약업신문사 2000 의약정보 Vol.2000 No.2

Novel Regulatory Mechamism of Carcinogenesis and Anti-Carcinogenesis in HPV-Mediated Cervical Cancer

Um, Soo Jong 가톨릭 의과학연구원 1999 가톨릭 의과학연구원 국제학술대회 Vol.3 No.-

부체형 HPV-16DNA 를 함유한 자궁경부암에서 HPV-16URR 유전자의 변이에 따른 전사 활성도 증가

김찬주,김은주,엄수종,박종섭,남궁성은,이해남,나종구,김승조 대한부인종양 콜포스코피학회 1998 Journal of Gynecologic Oncology Vol.9 No.4

HPV E2 protein is known to act as a negative regulator of transcription and the disruption of E2 open reading frame by HPV integration can release suppression of E6 and E7 mRNA expression, resulting in uncontrolled cellular growth and malignant transformation by inactivating tumor suppressor gene products (p53, pRb). YY1 mutation of HPV URR has been suggested as one of indicator that explains development of cervical neoplasia by episomal type of HPV. To extend this hypothesis, we examined whether mutation(s) in specific sites of HPV URR is functionally related to the invasiveness of cervical neoplasia and the physical status of HPV DNA. The URR sequences were obtained by PCR amplification of HPV-16 genome from CIN and invasive cancer patients, cloned into pUC18 for sequencing, and into pBLCAT8+ for functional CAT assay. Our previous data classified HPV-infected patients into three groups: 3 cancer cases carrying episomal HPV DNA; 12 cancer cases carrying integrated HPV DNA; 12 CIN cases carrying episomal HPV DNA. The specific variants in HPV-16 URR were found in Korean women: GA transition at nt 7520 (100%, 27/27), AC transition at nt 7729 (70%; 19/27), and GA transition at nt 7841 (78%; 21/27). Selective mutations were observed at the YY1-binding sites of HPV-16 URR in the 3 patients with invasive cervical cancer, who having the episomal forms of HPV-16 DNA: AC transition at nt 7484 and GA transition at nt 7488 (YY1-binding site 2; from 7481 to 7489). Additionally, CT transition at nt 7785 (YY1-binding site 3; from 7781 to 7790) was found from 2 of 3 patients. No YY1 site mutations were detected in the 12 CIN patients and in the HPV-integrated invasive cancer patients. To determine whether these mutations have effect on the expression of HPV E6/E7 genes driven by URR, the transient transfection assay was employed using URR-CAT reporter plasmid. The relative activities of three URR mutants from episomal HPV-16 DNA of cervical cancers were 2- to 4-fold higher than that of HPV-16 URR prototype. In contrast, the URRs from integrated HPV-16 DNA in cervical cancer and from episomal HPV-16 DNA in CIN, where no mutation of the YY1-binding site was detected, showed similar levels of promoter activity to that of URR prototype. Our results support the hypothesis that the mutation at YY1 binding site is functionally related to the development of cervical neoplasia caused by episomal HPV-16 DNA in Korean cervical cancer patient. Thus, mutation in YY1 site of episomal HPV-16 URR may play a role of HPV integration in the progression of cervical cancer.

외인성 에스트로젠에 의한 자궁경부암 세포의 성장과 HPV 유전자 조절

이진우,김찬주,박태철,엄수종,박종섭,이준모,남궁성은,김승조 대한부인종양 콜포스코피학회 1998 Journal of Gynecologic Oncology Vol.9 No.4

Backgrounds: Human papillomavirus (HPV) infection is known as the major causative phenomenon in the development of cervical cancer. E6 and E7 proteins of oncogenic HPV types can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, long term use of oral contraceptives may be one of the risk factor for cervical cancer. Purpose: Investigation of estrogenic and anti-estrogenic effects on the proliferation of cervical cancer cells and gene expression of HPV under the regulation of HPV upstream regulatory region (URR) would help to explain the role of estradiol in HPV-associated pathogenesis of cervical cancer. Methods: Cervical cancer cells (HeLa, CaSki and C33A) were cultured in vitro in the presence of 17 β-estradiol or tamoxifen and the numbers of cells were directly counted to observe the growth stimulatory or suppressive effect of the treatment. The correlation between the growth regulatory effect and HPV E6/E7 gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient co-transfection assays, which were conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. Supplemental effect of estrogen receptor on the URR promoter activity was also evaluated. To analyze the growth suppressive function at the higher concentration of estradiol or tamoxifen in HeLa cells, DNA fragmentation assay was performed. Results: The proliferation of HeLa and CaSki cells was stimulated by estradiol at the concentration of physiological level (≤1 X 10-6M), reaching maximal growth at 0.5 X 10-6M. At concentration of 0.1 X 10-6M, tamoxifen also stimulated the proliferation of HeLa and CaSki cells. In contrast to HPV-positive cervical cells, C33A cells were not influenced to cell proliferation by addition of estradiol at the physiological level, indicating that HPV might play role in growth stimulatory effect of estrogen or tamoxifen. Interestingly, the proliferation of (5 and 10 X 10-6M), whereas those of CaSki and C33A cellswere not responded and littleHe La cells was totally suppressed by estradiol and tamoxifen at the higher concentration suppressed at the concentration, respectively. The levels of HPV-18 E6 and E7 mRNA were significantly increased after treatment of 0.5 X 10-6M estradiol as determined by RT-PCR. Furthermore, transient transfection experiments using the URR-CAT reporter plasmid indicatedthat the increased expression of HPV E6/E7 genes was related with the growth stimulatory effect of estradiol and tamoxifen. In addition, co-transfection of estrogen receptor (ER) leads to an over 4-fold increase in CAT activity after treatment of estradiol or tamoxifen with 0.5 X 10-6M. When estradiol or tamoxifen was treated at the concentration over 5 X 10-6M for 96 hr, a typical DNA ladder, a indicative of apoptosis, was observed in HeLa cells. However, DNA ladder was not detected in C33A cells of which growth was some suppressed under same concentration of estradiol. Conclusion: At the physiological levels, estradiol stimulated the growth of HPV-positive cervical cancer cells and tamoxifen also did at the concentration of 0.1 X 10-6M. The increased expression of HPV E6/E7 at the physiologic levels appeared to be related with the growth stimulation of HPV-positive cervical cancer cells. Growth suppression observed at the higher concentration (5 and 10 X 10-6M) might be a indicative of apoptosis shown by DNA fragmentation assay in HeLa cells. Taken together, these data suggested that the concentration of estradiol (≤1 X 10-6M) could be a risk-factor in HPV-mediated cerivcal carcinogenesis.

Ursolic acid에 의한 자궁 경부암 세포주에서 항증식, 항바이러스 작용 기전 분석

이근호,임은경,윤주희,엄수종,남궁성은,박종섭 대한부인종양 콜포스코피학회 2003 Journal of Gynecologic Oncology Vol.14 No.4

목적 : 동양의 전통적인 약제로 사용되고 있는 ursolic acid는 항암, 항염증 효과가 있다고 알려져 있다. 본 연구는 ursolic acid의 자궁경부암 세포주에 대한 증식 억제와 HPV에 관련된 항바이러스 효과를 관찰하고자 하였다. 연구 방법 : Ursolic acid와 Dexamethasone을 자궁경부암 세포주에 처리한 후 세포 증식 억제 효과를 관찰하기 위하여 MTT assay를 시행하였으며, DNA fragmentation, DAPI staining과 FACS 분석을 통해 apoptosis의 특징을 관찰하였으며, apoptosis와 연관된 단백질들로 western blotting을 시행하였다. 또한, 항바이러스 효과를 관찰하기 위하여 HPV E6, E7의 발현 정도를 RT-PCR로 분석하였다. 결과 : Ursolic acid를 HPV가 함유된 자궁경부암 세포주(HeLa, CaSki, SiHa)에 투여하면 농도와 시간에 의존적으로 비례하여 세포증식이 억제되었으나, HPV를 함유하지 않은 자궁경부암 세포주(C33A)에서는 세포 증식억제 효과는 없었다. HeLa 세포주에서 ursolic acid의 농도를 증가시킴에 따라 apoptosis의 특징을 보여주는 DNA fragmentation, DAPI staining, FACS 분석 소견이 뚜렷하게 나타났으며, westem blottiong을 통하여 Fsa 단백질의 발현이 유도되고, caspase-8, caspase-3, PARP 단백질이 절단됨을 관찰할 수 있었다. HeLa 세포에 ursolic acid를 처리함에 따라 HPV-18 E6, E7 유전자 발현이 감소되었으나, p53과 Rb 단백질의 변화는 관찰할 수 없었다. 결론 : 자궁경부암 세포에 ursolic acid를 투여하면 세포증식 억제가 apoptosis에 의하여 일어나며, HPV E6/E7의 발현저하로 인한 항바이러스 효과도 기여할 것으로 사료된다. 이 연구 결과는 ursolic acid에 의한 항증식, 항바이러스 기전을 이해하는데 도움을 주었으며, 효과적인 약제로서의 사용 가능성을 제시하였다. Objective : Ursolic acid which has been used as a herb medication, was reported for anti-cancer and anti-inflammatory effects. We tried to analyze the anti-proliferative and anti-viral effects of ursolic acid in Human papillomavirus (HPV) associated cervical carcinoma cell lines. Methods : We treated ursolic acid or dexamethasone to cervical carcinoma cell lines. We carried out MTT assay to observe the anti-proliferative effects and tried to find out the characteristics of apoptosis by DNA fragmentation, DAPI staining and FACS analysis. By means of westem blotting, we investigated proteins related with apoptosis. After treating of ursolic acid, we used RT-PCR for the expression of HPV E6 and E7 gene to observe the anti-viral effects. Through proteomics analysis including two-dimensional electrophorosis (2DE) and matrix-associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we found the interaction between cell cycle regulatory factor and apoptosis related protein, and also demonstrated anti-cancer and anti-viral mechanism. Results : While the growth of HPV-positive cervical carcinoma cell lines (HeLa, CaSki, SiHa) is suppressed by ursolic acid in a dose-and time-dependent manner, that of HPV-negative cervical carcinoma cell line (C33A) did not have any inhibitory effects on proliferation. As we increased the concentrations of ursolic acid in HeLa cell, the typical characteristics of apoptosis were noted in DNA fragmentation, DAPI staining and FACS analysis, The expression of Fas protein was induced and caspase-8, caspase-3 and PARP proteins changed into cleavaged forms after treating ursolic acid. HPV-18 E6/E7 gene expression decreased after treating ursolic acid in HeLa cells, but the levels of p53 and Rb proteins did not changed. Total 30 spots were detected by proteomics analtsis, along with 20 up-regulated, 6 down-regulated and 4 unknown protein were founded. Conclusion : These results suggest that ursolic acid inhibit the cell proliferation through apoptosis in HPV-associated cervical carcinoma cell lines. The apoptotic mechanism of ursolic acid in HeLa cells might be through the signal pathway "Fas→caspase-8→PARP". In addition, the antiviral effect of ursolic acid through suppression of HPV E6/E7 gene expression would contribute to growth inhibition in HeLa cells. These results suggest that ursolic acid could be useful for an effective anticancer drug in treatment of HPV-associated cervical neoplasia.