ヒトglioma細胞亜群の分離とその継代株の検討—コロニー形成能とBCNU感受性試験

CLONOGENICITY AND BCNU RESPONSE OF SUBPOPULATIONS OF HUMAN GLIOMA CELLS SORTED ACCORDING TO DNA CONTENT

　抄録　Flow cytometerによりヒトglioma細胞のDNA量分析を行うとdiploid以外にaneuploidやheteroploidの細胞を包含することが知られている。今回DNA量の差でglioma細胞亜群を分離する実験を行い，gliomaを細胞レベルから分析した。各亜群ごとにコロニー形成能とBCNU感受性を調べ，さらに亜群細胞の継代培養によるDNAヒストグラムとBCNU感受性の変化を追跡した。ヒトglioma由来8細胞株を対象とし　chromomycin A₃染色によるDNA量分析およびHoechst 33342 vital stainingによる細胞亜群の分離をFACS-III （B-D）で行い，BCNU感受性試験はclonogenic cell assayで評価した。　DNAヒストグラム上3個のピークを持つ細胞株からピークごとに採取した細胞亜群はいずれもコロニー形成能を持っていた。ピーク1のコロニー形成能が低い細胞株では継代によりピーク1の相対細胞数が減少し，これは分離後継代した亜群細胞にも受けつがれた。分離後継代した亜群細胞のDNA量分析およびBCNU感受性試験により，DNA量およびBCNU感受性の異なった2個の細胞亜群から構成されるヒトgliomaの存在が明らかとなった。

Malignant gliomas, especially glioblastoma mul-tiforme, are composed of cells that contain various amounts of DNA. We characterized cells dissoci-ated from biopsy specimen of human gliomas by sorting vital cell populations on the basis of DNA content, determined the sensitivity of sorted cells to BCNU, and did the follow-up study using subpopulations cultivated for a long time after sorting on both the DNA histogram and the BCNU response.

Seven malignant human gliomas carried in monolayer culture which had three peaks on the DNA histogram were used for this study. To obtain each DNA distribution histogram, cells were stained with chromomycin A 3 and were analyzed with a modified FACS III flow cytometer. Vitai DNA was stained with Hoechst 33342 by means of a modification of the procedure of Jovin et al, which involved negligible toxicity to cells. Cells were sorted on FACS III at each peak on the DNA histogram. The colony forming efficiency (CFE) of each population was determined after sorting and surviving fraction was determined after exposure to graded doses (2.5-10 μg/ml×2 hr) of BCNU. Six subpopulations derived from two human glioma cells were cultivated for a long time and reevaluated by both the DNA histogram and the sensitivity to BCNU.

The CFEs for each cell population of three peaks on DNA histograms of human glioma cells were similar, except that the CFE for the far left peak (peak 1) in each histogram appeared to be slightly lower in four specimens. In three out of those tumors, the relative number of cells included in the peak 1 decreased after multiplepassages in accordance with the lower CFE values. Subpopulations of a tumor cultivated for a long time after sorting preserved this tendency of losing peak 1 population.

There was a variable response with a mag-nitude of close to 1 log when each population of human glioma cells was studied of its sensitivity to BCNU immediately after sorting. By cultivation of subpopulations, we found that a tumor con-tained two distinct subpopulations, which had different sensitivities to BCNU. This lack of uni-formity in the response of cells within a tumor may contribute to the appearance of clinical re-sistance to antitumor agents.

Supported by Grant PDT-159 from the Ameri-can Cancer Society and Grant CA-13525 from the NIH.