Molecular analysis of the differentiation associated events and screening of new differentiation factors in F9 teratocarcinoma stem cells

Abstract

F9 embryonal carcinoma 세포는 정상적인 세포배양상태에서는 자발적인 분화를 일으키지 않으나 retinoic acid와 dibutyryl cyclic AMP를 처리하면 분화가 유도된다. F9 세포의 분화는 비가역적이며 형태적인 변화와 더불어 세포와 기질 단백질이 증가하는 것과 같은 생화학적인 변화가 일어난다. 그러므로 이 F9 embryonal carcinoma 세포를 모델시스템으로 하여 분화의 기작 및 새로운 분화인자를 검색하는 것이 가능하다. 이 논문의 목적은 retinoic acid가 F9 embryonal carcinoma 세포의 분화에 미치는 영향을 알아보고 먼저 retinoic acid와 dibutyryl cyclic AMP에 의해 분화가 유도된 F9세포에서의 세포와 기질단백질 및 5종류의 암유전자의 발현을 알아본 다음 세포외 기질 단백질이 F9 embryonal carcinoma 세포의 분화에 미치는 영향을 연구하고자 한다. 또한 retinoic acid와 같이 F9세포의 분화를 일으키는 새로운 분화인자들을 검색한 다음 이 새로운 분화인자들의 작용기작을 연구한 결과 retinoic acid를 처리한 F9세포에서 형태학적인 변화, 성장 중지 및 laminin, type IV collagen과 같은 세포외기질 단백질의 발현이 증가하고 fibronectin 및 c-myc, c-Ki-ras, c-erb B와 같은 암유전자의 발현은 감소하였다. 또한 c-raf의 발현은 변하지 않았다. 한편 laminin 및 collagen을 처리한 flask에서 배양한 세포는 retinoic acid를 처리하여 분화시킨 F9세포와 같은 형태를 띄었다. retinoic acid와 같은 분화유도를 가지는 새로운 분화유도인자를 검색한 결과 인삼에서 추출한 saponin, 비파에서 추출한 ursolic acid, oleanioc acid 및 감초에서 추출한 glycyrrhizine이 분화유도인자들의 효과를 나타내었으며 구조 및 southwestern analysis, competition assay 결과 이 인자들은 steroid receptor에 결합해 그 작용을 나타내는 것으로 보여진다. ; F9 teratocarcinoma stem cells show very low spontaneous differentiation under normal culture condition. However, F9 cells were induced to differentiate in the presence of retinoic acid and diutyryl cyclic AMP. The differentiation response of F9 cells is irreversible and is accompanied by morphological alteration and biochemical change such as increase expression of extracellular matrix protein. The aim of this study is to investigate the mechanism of differentiation and to screen new differentiation factors using F9 teratocarcinoma stem cells as a model system. The first part of this work examined the patterns of expression of extracellular matrix components and 5 oncogenes during the differentiation of F9 cells induced by retinoic acid and dbc AMP. In the second part, the effect of extracellular matrix components on the differentiation of F9 EC cells was identified. In the third part, an attempt was made to determine new differentiation factors which show differentiation inducing activities characterized by the alteration of morphology and the expression of differentiation specific genes in F9 cells. Finally an attempt was made to clarify the differentiation inducing mechanism of new differentiation factors. According to results, F9 cells cultured in the presence of retinoic acid became round and stopped growth. The mRNA level of extracellular matrix protein gene, such as laminin and type IV collagen, was increased markedly the expression of fibronectin and 3 oncogenes, c-Ki-ras, c-myc and c-erb B, were decreased and the mRNA level of c-fos was increased. The expression of c-raf was not changed in F9 cells was not changed in F9 cells cultured in the medium containing retinoic acid. When f9 cells cultured on the substratum of laminin and type IV collagen became round as the differentiation F9 cells induced by retinoic acid and dbcAMP. In the process of screening new differentiation factor, ginseng saponin, dexamethasone, ursolic acid, oleanoic acid and glycyrrhizine were shown to have differentiation inducing effect. F9 cells exposured to ginseng saponin, dexamethasone, ursolic acid or oleanoic acid became round as the retinoic acid-treated F9 cells. By examining the structure of ginseng saponin, ursolic acid, oleanoic acid and glycyrrhizine, it was suggested that those factors exert those differentiation inducing function by binding steroid receptor. According to the southwestern analysis and competition assay. It was confirmed that those differentiation factors induce differentiation via steroid receptor.