9L-グリオーマ細胞におよぼすcyclic AMPの影響—Flow cytometryによる生長解析

FLOW CYTOMETRIC ANALYSIS FOR EFFECT OF cAMP ON CELL CYCLE OF 9L RAT GLIOMA, IN VITRO

　抄録　Cyclic AMP （cAMP）はホルモンのsecond messengerとして考えられ，細胞の分化と関係しているといわれている。特に増殖能に関しては，正常の細胞よりも悪性の腫瘍細胞に対して，より抑制が強いという報告がされている。我々も既にヒト悪性脳腫瘍の初代培養細胞について同様の増殖抑制を認め報告して来たが，今回9L cell lineを用いて詳細にこのことを検討してみた。方法はBrdUモノクロナール抗体を用いたflow cytometry分析によって行った。その結果cAMPはDNA合成面に直接関与しておらず，細胞回転上からみると，G₂期の延長とG₁期での細胞回転の停止であることを明確にできた。さらにacridine orangeによる二重染色にて，細胞はRNA量の含有量の少ないG₁期に集積していることが分かり，cyclic AMPの細胞回転における関与が，静止細胞（G₀）の増加であり，単なるG₁期での停止でないことが明らかになった。これらの事実は悪性脳腫瘍の分化に関する研究に，またこの薬剤を治療に応用する場合において，重要な情報と思われる。

Perturbed cell kinetics in 9 L rat brain tumor treated with dibutyryl cyclic AMP (DBcAMP) and theophylline were studied. The methods in-clude a procedure for simultaneous flow cytometric measurements of cellular DNA and amount of bromodeoxyuridine (BrdU) incorporated into cel-lular DNA and a procedure for flow cytometric measurements of cellular DNA and RNA. Pro-pidium iodide and monoclonal antibody against BrdU were used as a fluorescenct probe for total cellular DNA and for BrdU incorporated into cellular DNA, respectively. For analysis of cel-lular DNA and RNA, acridine orange stainning was also applied.

The results were as following; cells in S and G₂ phase at the time of drug administration (1 mM, each) can undergo mitosis even though a considerable prolongation of G₂ phase was appar-ent. However, cells in G₁ at the time of drug administraion were arrested in that phase, whereas those cells in S or G₂ were able to complete one mitosis before becomimg arrest in the G₁ phase. These results have been inferred from the study by autographic method with ³⁾H-thymidine.

However, using these new methods, these were clearly demonstrated and furthermore, flow cyto-metric analysis with acridine orange revealed that DBcAMP induced 9 L cells into resting posi-tion (G₀ cells).