Cell cycle distribution of chronically hypoxic cells and determination of the clonogenic potential of cells accumulated in G2 + M phases after irradiation of a solid tumor in vivo.
Cell cycle distribution of chronically hypoxic cells and determination of the clonogenic potential of cells accumulated in G2 + M phases after irradiation of a solid tumor in vivo.
复制标题
体内实体瘤照射后慢性缺氧细胞的细胞周期分布以及 G2M 期积累的细胞克隆潜力的测定。
作者：
M. Pallavicini;M. Lalande;R. Miller;R. Hill
从细胞周期扰动的流式细胞术分析中获得的信息受到DNA分布中区分克隆性细胞和非克隆性细胞的问题的限制。Hoechst 33342是一种dna特异性染色剂，据报道对细胞活力的影响很小。在这些研究中，它被用于研究根据DNA含量分类的未照射和照射过的实体瘤细胞的克隆原性。通过体外实验确定未辐照KHT肉瘤细胞的克隆生成潜力，g1期、S期和g2 + M期细胞的克隆生成潜力无显著差异。KHT肿瘤细胞为四倍体，在肿瘤中检测到“正常二倍体群体”。二倍体群体的电镀效率极低。大剂量（1700拉德）照射原位肿瘤后，存活的缺氧细胞85%处于g1期，9.6和5.6%分别处于S期和g2 + M期。通过对米霉素染色细胞的流式细胞术分析，记录了辐射诱导的KHT肿瘤细胞周期扰动的时间过程。照射10小时后，g2 + M细胞百分比增加2.5倍。48小时时仍然存在明显的扰动。用Hoechst 33342获得了类似的结果，并在辐射后10小时测定了包含DNA分布的细胞的克隆潜能。在g1期存活的克隆原细胞约占77%，而在g2 + M期存活的克隆原细胞仅占23%，尽管g2 + M期肿瘤细胞总数大量增加。这些数据证明了流式细胞术分析和从实体肿瘤中获得的细胞分选在放射生物学现象研究中的应用。
Abstract Information obtained from flow cytometric analysis of cell cycle perturbations has been limited by the problem of distinguishing clonogenic from nonclonogenic cells in a DNA distribution. Hoechst 33342 is a DNA-specific stain reported to have minimal effects on cell viability. It was used in these studies to investigate the clonogenicity of unirradiated and irradiated solid tumor cells sorted according to their DNA content. The clonogenic potential of unirradiated KHT sarcoma cells was determined with an in vitro assay and was not significantly different for cells in G 1 , S, and G 2 + M phases. The KHT tumor cells are tetraploid, and a “normal diploid population” was detected in the tumor. The plating efficiency of the diploid population was minimal. After a large dose of radiation (1700 rads) to the in situ tumor, 85% of the surviving hypoxic cells were found in G 1 phase, and 9.6 and 5.6% were in S and G 2 + M phases, respectively. The temporal course of radiation-induced cell cycle perturbations in the KHT tumor was documented by flow cytometric analysis of mithramycin-stained cells. A 2.5-fold increase in the percentage of cells in G 2 + M was observed 10 hr after radiation. Significant perturbations still existed at 48 hr. Similar results were obtained with Hoechst 33342, and the clonogenic potential of the cells comprising the DNA distribution was determined 10 hr after radiation. Approximately 77% of the surviving clonogenic cells were in G 1 phase, and only 23% were found in G 2 + M in spite of the large increase in the total number of tumor cells in these phases. These data demonstrate applications of flow cytometric analysis and sorting of cells obtained from solid tumors to the investigations of radiobiological phenomena.