培養ラット胎児アストログリア細胞のENU発癌—N-Methyl-N‐nitrosourea（ENU）誘発変異細胞出現とその細胞生物学的検討

In Vitro ENU-induced Carcinogenesis of Rat Fetal Astroglia : Biological Character of Mutant Glial Cell

　グリア細胞の培養系でのENU発癌の可否，および早期の腫瘍性変化を観察することを目的として，ラット胎児アストログリアをENU処理後，長期継代培養した。その結果，高濃度（150μg／ml）ENU処理後，少なくとも40日以後にcontact inhibitionの消失したコロニーが出現し，この中から4つの変異細胞株を樹立した。これらの4株はすべてGFAP陽性でアストログリア由来と考えられた。4株の細胞はいずれも低血清（2％）培養液中で約30％のコロニー形成能を示し，またヌードマウス皮下移植により腫瘍を形成した。変異型のみを認識する抗p53モノクローナル抗体を用いて行った免疫染色の結果では，初代培養細胞およびENU未処理細胞は陰性であったが，変異細胞はすべて陽性であり，p53の変異がラット胎児アストログリアの腫瘍化に強く関与していることが示唆された。本研究で樹立した変異細胞株はグリアの腫瘍性変化を経時的に観察するために有用であると考えられる。

To investigate glial carcinogenesis in vitro, fetalat brain cells were cultured and exposed to ENU(apploximately 200 μg/ml). The cells were pas-saged weekly thereafter. Morphological changes were observed under the phase contrast microscope. When mutant colonies where the cells lost contact inhibition and grew in a multilayer fashion appear-ed, the cells were cloned. To assess the biological characters of cells, expression of GFAP, vimentin, A2B5 and p53 product were determined by immuno-histochemistry and flow cytometry. Tumor formingability of the cells was evaluated by both colony forming efficiency in low serum medium (LSM ; 2 % FBS, 300 cells/100 mm dish) and trans-plantability to nude mice. Both primary cultured and ENU-treated cells were positive for GFAP and vimentin, but population of A2B5 positive cells was less than 5 %, thus indicating that these cells were astroglial in origin. The mutant colonies appeared 7 weeks after ENU treatment. These cells grew rapid-ly with cell doubling time ranging between 18 to 26 hours, while non-ENU-treated astroglias had a longer cell doubling time (48 to 55 hours). The cloned mutant glial cell lines formed large colonies in LSM (efficiency 20~40 %) , but astroglial cells did not. The mutant astroglial cells also developed tumors in nude mice. p53 protein was never detected in normal astroglia, however, some glial cells treat-ed by ENU abruptly became p53 positive after several passages. These p53 positive cells formed stratified colonies thereafter. These results indicate that mutant astroglial cells can be induced by a single dose of ENU in vitro. As the p53 protein which was detected immunohistochemically has been thought to be mutant, it was suggested that the mutation of p53 may be one of the early major steps in glial carcinogenesis.