McGinn C J, Miller E M, Lindstrom M J, Kunugi K A, Johnston P G, Kinsella T J
Department of Human Oncology, University of Wisconsin Medical School, Madison 53792.
Int J Radiat Oncol Biol Phys. 1994 Nov 15;30(4):851-9. doi: 10.1016/0360-3016(94)90360-3.
Radiosensitization has previously been demonstrated in a human colon cancer cell line (HT-29) following a 2 h exposure to low, clinically relevant concentrations (0.05-0.5 microM) of fluorodeoxyuridine (FdUrd) (15). The sensitizer enhancement ratio value (measured at 10% survival) plateaued at approximately 1.7 between 16 and 32 h following removal of drug. Parallel studies investigating the effect of FdUrd on the distribution of cells throughout the cell cycle found that the percentage of cells in early S-phase increased to approximately 70% during the same period that maximal radiosensitization was noted. As a follow-up to these findings, experiments have been designed to investigate the contribution of this early S-phase delay to radiosensitization.
Synchronized populations of HT-29 cells have been obtained with three separate techniques. Two involve the induction of a reversible metaphase arrest (with high pressure N2O or colcemid) followed by a shakeoff of mitotic cells. The third uses a plant amino acid, mimosine, to induce a reversible block at the G1/S boundary. Flow cytometry was used to analyze the degree of synchrony based on bromodeoxyuridine (BrdUrd) uptake and propidium iodide (PI) staining. Radiation survival curves were obtained on these synchronized populations to investigate changes in radiosensitivity through the cell cycle. Additionally, levels of thymidylate synthase (TS), the primary target of FdUrd cytotoxicity, were measured in each phase of the cell cycle using the TS 106 monoclonal antibody against human TS.
Synchronization with mitotic shakeoff produced relatively pure populations of cells in G1; however, the degree of synchrony in early S-phase was limited both by cells remaining in G1 and by cells progressing into late S-phase. These techniques failed to reveal increased radiosensitivity in early S-phase at 10% survival. An 18 h exposure to mimosine resulted in populations that more closely resembled the early S-phase enrichment following FdUrd exposure and revealed increased radiosensitivity during early S-phase. TS levels were noted to be only 1.3 times higher in S phase than in G0/G1.
Radiation survival data from cells synchronized with mitotic shakeoff techniques suggest that early S-phase delay is unlikely to be the primary mechanism of FdUrd radiosensitization. In contrast, the increased sensitivity seen in early S-phase with mimosine synchronized cells is similar to that seen with FdUrd. Although confounding biochemical pertubations cannot be ruled out, these data continue to suggest an association between early S-phase enrichment and radiosensitization. The significance of TS inhibition as a mechanism of FdUrd radiosensitization remains unclear.
先前已证明，人结肠癌细胞系（HT - 29）在暴露于低的、临床相关浓度（0.05 - 0.5微摩尔）的氟脱氧尿苷（FdUrd）2小时后会出现放射增敏作用（15）。在去除药物后的16至32小时之间，增敏剂增强比值（在10%存活率时测量）稳定在约1.7。平行研究调查FdUrd对整个细胞周期中细胞分布的影响发现，在观察到最大放射增敏作用的同一时期，早S期细胞的百分比增加到约70%。作为这些发现的后续研究，已设计实验来研究这种早S期延迟对放射增敏的作用。
已通过三种不同技术获得了HT - 29细胞的同步化群体。两种技术涉及诱导可逆的中期阻滞（使用高压N₂O或秋水仙酰胺），随后抖落有丝分裂细胞。第三种技术使用植物氨基酸含羞草碱，在G₁/S边界诱导可逆阻滞。基于溴脱氧尿苷（BrdUrd）摄取和碘化丙啶（PI）染色，使用流式细胞术分析同步化程度。在这些同步化群体上获得放射存活曲线，以研究整个细胞周期中放射敏感性的变化。此外，使用针对人胸苷酸合成酶（TS）的TS 106单克隆抗体，在细胞周期的每个阶段测量TS的水平，TS是FdUrd细胞毒性的主要靶点。
通过有丝分裂抖落进行同步化产生了相对纯合的G₁期细胞群体；然而，早S期的同步化程度受到仍处于G₁期的细胞以及进入晚S期的细胞的限制。这些技术未能揭示在10%存活率时早S期放射敏感性增加。暴露于含羞草碱18小时产生的细胞群体更类似于FdUrd暴露后的早S期富集，并显示出早S期放射敏感性增加。注意到S期的TS水平仅比G₀/G₁期高1.3倍。
来自通过有丝分裂抖落技术同步化的细胞的放射存活数据表明，早S期延迟不太可能是FdUrd放射增敏的主要机制。相比之下，含羞草碱同步化的细胞在早S期观察到增加的敏感性，这与FdUrd的情况相似。尽管不能排除混杂的生化扰动，但这些数据继续表明早S期富集与放射增敏之间存在关联。TS抑制作为FdUrd放射增敏机制的意义仍不清楚。
