Grégoire V, Van N T, Stephens L C, Brock W A, Milas L, Plunkett W, Hittelman W N
Department of Clinical Investigation, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Cancer Res. 1994 Dec 1;54(23):6201-9.
We have previously reported that fludarabine, an adenine nucleoside analogue, significantly enhances radiation-induced tumor regrowth delay and local cure in several mouse tumors. Although fludarabine potentiated tumor regrowth delay at various times from -36 h to +6 h in a SA-NH mouse sarcoma model, the greatest enhancement was observed when fludarabine was administered 24 h before irradiation. The purpose of this study was to understand the basis for in vivo enhancement of radiation efficacy by fludarabine. To examine the effect of fludarabine on DNA synthesis and cell cycle progression, tumor-bearing mice were given fludarabine by an i.p. route and then bromodeoxyuridine at various times up to 36 h, followed 0.5 h later by tumor harvest. Two-parameter flow cytometry analysis of the tumor cells using an anti-bromodeoxyuridine antibody demonstrated that an 800-mg/kg fludarabine dose stops DNA synthesis within 3 h with recovery starting at 12 h. By 24 h after fludarabine treatment, a synchronized wave of cycling tumor cells appeared in G2-M phase. The degree of DNA synthesis shutdown and the timing of the reinitiation of DNA synthesis and cell cycle progression were all fludarabine dose dependent. Interestingly, DNA synthesis reinitiated only at the G1-S boundary; cells in the S phase at the time of fludarabine administration appeared to disappear from the tumor population. To confirm these observations more directly, we pretreated tumor-bearing mice i.p. with chlorodeoxyuridine to mark the cells in the S phase, gave them fludarabine 0.5 h later, and then gave them iododeoxyuridine 0.5 h before tumor harvest. Flow cytometry analysis using antibodies specific for chlorodeoxyuridine- and iododeoxyuridined-labeled cells confirmed that cells in the S phase at the time of fludarabine administration never reinitiated DNA synthesis and disappeared from the tumor population. Immunohistological analysis of tumor sections obtained after fludarabine administration demonstrated that prelabeled S-phase cells took on an apoptotic appearance and gradually disappeared from the tumors. An in situ DNA end labeling assay demonstrated DNA fragmentation in these morphologically apoptotic cells. These results suggest that the mechanism of fludarabine enhancement of radiation response involves induced S-phase cell loss through an apoptotic pathway and subsequent synchronization of the remaining cells to a more radiosensitive cell cycle phase at the time of irradiation.
我们之前曾报道，腺嘌呤核苷类似物氟达拉滨可显著增强多种小鼠肿瘤的辐射诱导肿瘤再生长延迟及局部治愈效果。在SA-NH小鼠肉瘤模型中，尽管氟达拉滨在从-36小时至+6小时的不同时间点均能增强肿瘤再生长延迟，但在照射前24小时给予氟达拉滨时观察到最大程度的增强。本研究的目的是了解氟达拉滨在体内增强辐射疗效的基础。为了研究氟达拉滨对DNA合成及细胞周期进程的影响，给荷瘤小鼠经腹腔注射氟达拉滨，然后在长达36小时的不同时间点给予溴脱氧尿苷，0.5小时后收获肿瘤。使用抗溴脱氧尿苷抗体对肿瘤细胞进行双参数流式细胞术分析表明，800mg/kg的氟达拉滨剂量在3小时内可使DNA合成停止，12小时开始恢复。氟达拉滨治疗后24小时，出现了同步的处于G2-M期的循环肿瘤细胞波。DNA合成关闭的程度以及DNA合成和细胞周期进程重新启动的时间均呈氟达拉滨剂量依赖性。有趣的是，DNA合成仅在G1-S边界重新启动；在给予氟达拉滨时处于S期的细胞似乎从肿瘤群体中消失。为了更直接地证实这些观察结果，我们经腹腔给荷瘤小鼠预先注射氯脱氧尿苷以标记处于S期的细胞，0.5小时后给予它们氟达拉滨，然后在收获肿瘤前0.5小时给予它们碘脱氧尿苷。使用针对氯脱氧尿苷和碘脱氧尿苷标记细胞的特异性抗体进行流式细胞术分析证实，在给予氟达拉滨时处于S期的细胞从未重新启动DNA合成并从肿瘤群体中消失。对给予氟达拉滨后获得的肿瘤切片进行免疫组织学分析表明，预先标记的S期细胞呈现凋亡外观并逐渐从肿瘤中消失。原位DNA末端标记试验证明这些形态学上凋亡的细胞中存在DNA片段化。这些结果表明，氟达拉滨增强辐射反应的机制涉及通过凋亡途径诱导S期细胞丢失，以及随后在照射时将剩余细胞同步到更具放射敏感性的细胞周期阶段。
