Rodriguez A, Alpen E L, Mendonca M, DeGuzman R J
Biology and Medicine Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Radiat Res. 1988 Jun;114(3):515-27.
Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.
与单层培养的相同细胞相比，以多细胞肿瘤球体形式生长的细胞表现出放射抗性。对以球体形式生长的9L细胞和汇合单层细胞的潜在致死性损伤（PLD）修复及其动力学进行了比较。照射后立即接种的细胞的存活曲线显示，与平台期单层细胞相比，球体培养具有典型的放射抗性。球体细胞存活的剂量修正因子为1.44。在正常磷酸盐缓冲盐水（PBS）、条件培养基或PBS中的0.5 M NaCl中进行照射后孵育，可减少两种培养条件下放射敏感性的差异。在PBS或条件培养基中进行照射后处理可促进潜在致死性损伤的修复，而0.5 M NaCl可阻止PLD的去除并使损伤固定，从而导致存活率降低。高渗NaCl处理后球体细胞和单层细胞的存活率相同。NaCl处理对存活曲线的Do的降低幅度大于对肩部（Dq）的降低幅度。在PBS中进行照射后孵育，然后进行高渗NaCl处理后测量的PLD修复动力学，对于球体和平台期单层细胞是相同的。PLD修复动力学表明是一种双相现象。有一个初始的快速成分，修复半衰期为7.9分钟，还有一个缓慢成分，修复半衰期为56.6分钟。大部分损伤（59%）修复缓慢。由于球体和单层细胞的修复能力和动力学相同，因此不能据此解释球体的放射抗性。有证据表明，对于汇合单层细胞和蛋白酶处理解离后的球体细胞，从Go（非循环G1期细胞）状态恢复到增殖状态（再增殖）的时间可能是发生的PLD修复程度的最重要决定因素。生长曲线和流式细胞术细胞周期分析表明，球体细胞重新进入增殖状态有一个延迟期。由于在此期间接种效率保持高且不变，因此不能基于存在大量无潜在克隆能力的Go细胞来解释这种延迟。单层细胞的细胞周期进程在6 - 8小时开始，球体细胞在14 - 15小时开始。据推测，球体细胞较慢地重新进入循环期比快速增殖的单层细胞有更多时间进行修复。
