Cell cycle perturbations in acute myeloid leukemia samples following in vitro exposures to therapeutic agents
Cell cycle perturbations in acute myeloid leukemia samples following in vitro exposures to therapeutic agents
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DOI：
10.1016/s0145-2126(97)00174-4
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发表时间：
1998-03-01
影响因子：
2.7
通讯作者：
Appelbaum, FR
中科院分区：
文献类型：
--
作者：
Banker, DE;Groudine, M;Appelbaum, FR
细胞周期检查点确定了对损伤性治疗的阻滞、修复和凋亡反应的时间和强度。我们设计了流式细胞术测定，以测量细胞周期阻滞和细胞凋亡的急性髓性白血病（AML）样本在体外治疗相关的治疗药物，以便在功能上表征这些样本中的检查点，并询问检查点异常是否是常见的AML，并有助于治疗失败。我们在这里表明，细胞周期反应柔红霉素（DNR），阿糖胞苷（ARA-C）和γ射线照射（RAD）是可重复的处理剂和剂量依赖性和不同的骨髓细胞系。DNR处理在细胞周期的差距2和有丝分裂（G2/M）期和/或在差距1（G1）期诱导细胞蓄积，RAD也是如此，而ARA-C诱导在DNA合成（S）期或在G1期的蓄积。设计流式细胞术门以在分析原代骨髓细胞样本时排除淋巴细胞和成熟中性粒细胞。因此，来自正常供体的骨髓样品中的细胞亚群富含髓样成分，并用作正常髓样细胞对照。增殖细胞核抗原（PCNA）免疫染色用于进一步鉴定原代细胞样品中活跃分裂的细胞亚群。对AML样品进行了类似的分析，大多数显示出比正常骨髓细胞更低的DNA合成细胞周期（S）分数和更低的PCNA阳性分数，表明AML在这些培养条件下通常增殖性较低。具有高S期分数的例外AML样本具有与不良预后相关的细胞遗传学异常。大多数AML样本安装相对于正常骨髓细胞弱的细胞周期反应，而少数显示出强大的，代理特定的细胞周期停滞。这种非反应性并不简单地与较低的循环指数，无论是响应模式，也不与未处理的S期分数或与PCNA阳性分数的响应程度。细胞周期应答与临床参数（包括患者年龄、FAB分类或白色血细胞计数）、免疫表型特征（包括CD 34状态）和特异性细胞遗传学标志物均无关。这表明功能性细胞周期反应测定可以提供AML的独特诊断信息。这些测定也可能具有预后价值，因为ARAC诱导的G1期阻滞和DNR特异性G2/M期阻滞往往与未能实现临床缓解相关。此外，ARA-C后G1期阻滞和DNR治疗后G2/M期累积在先前已显示bcl-2表达更高免疫阳性的样本中往往更稳健。这些数据表明，bcl-2表达与特定的细胞周期反应的治疗剂的协会可能有助于bcl-2与AML的临床反应差。这些数据提供了进一步的实验室研究的基础，旨在检查特定的细胞周期阻滞作为治疗耐药的机制和前瞻性研究，旨在严格评估检查点功能的体外测定的预后效用。(C)1998爱思唯尔科技有限公司版权所有。
Cell cycle checkpoints establish the timing and strength of arrest, repair and apoptosis responses to damaging treatments. We designed flow cytometric assays to measure cell cycle arrest and apoptosis in acute myeloid leukemia (AML) samples treated in vitro with relevant therapeutic agents so as to functionally characterize checkpoints in these samples and to ask if checkpoint abnormalities are common in AML and contribute to therapeutic failures. We show here that cell cycle responses to daunomycin (DNR), cytosine arabinoside (ARA-C) and gamma irradiation (RAD) were reproducibly treatment agent-and dose-dependent and distinct in different myeloid cell lines. DNR treatments differentially induced cell accumulations in the gap 2 and mitosis (G2/M) phases of the cell cycle and/or in the gap 1 (G1) phase, as did RAD, while ARA-C induced accumulations in the DNA synthesis (S) phase or in the G1 phase. Flow cytometric gates were devised to exclude lymphocytes and mature neutrophils in analyses of primary myeloid cell samples. Cell subsets in bone marrow samples from normal donors were thus enriched for myeloid constituents and used as normal myeloid cell controls. Proliferating cell nuclear antigen (PCNA) immunostaining was used to further identify actively dividing cell subpopulations in primary cell samples. AML samples were similarly analyzed and the majority showed lower DNA synthesis cell cycle phase (S) fractions and lower PCNA-positive fractions than normal myeloid cells, suggesting that AMLs are generally less proliferative in these culture conditions. Exceptional AML samples with high S phase fractions had cytogenetic abnormalities associated with poor prognosis. Most AML samples mounted weak cell cycle responses relative to normal myeloid cells, while a minority showed robust, agent-specific cell cycle arrests. This non-responsiveness was not simply associated with lower cycling indices-neither the response patterns nor the degrees of response were correlated with untreated S phase fractions or with PCNA-positive fractions. Cell cycle responses were also not associated with clinical parameters including patient age, FAB class, or white blood cell count, nor with immunophenotypic features including CD34 status, nor with specific cytogenetic markers. This suggests that functional cell cycle response assays could provide unique diagnostic information in AML. These assays might also have prognostic value as ARAC induced G1 arrests and DNR-specific G2/M arrests tended to be associated with failure to achieve clinical remission. In addition, G1 arrests after ARA-C and G2/M accumulations after DNR treatments tended to be more robust in samples that had previously been shown to be more highly immunopositive for bcl-2 expression. This data suggests that the association of bcl-2 expression with particular cell cycle responses to therapeutic agents may contribute to the association of bcl-2 with poor clinical responses in AML. These data provide the basis for further laboratory studies aimed at examining specific cell cycle arrests as mechanisms of therapeutic resistance and prospective studies aimed at rigorously assessing the prognostic utility of in vitro assays of checkpoint function. (C) 1998 Elsevier Science Ltd. All rights reserved.