Walker A, Taylor S T, Hickman J A, Dive C
Cancer Research Campaign Molecular and Cellular Pharmacology Group, School of Biological Sciences, University of Manchester, United Kingdom.
Cancer Res. 1997 May 15;57(10):1939-45.
Bcl-2 suppresses drug-induced apoptosis in vitro, although in many cases, this results only in a delayed onset of cell death. In vivo survival signals from the extracellular environment may also contribute to drug resistance and may act with Bcl-2 to promote long-term cell survival. Ligation of CD40 on B-lymphocytes in germinal centers (GCs) can suppress apoptosis induced by calcium ionophore or anti-IgM in vitro. We asked whether a combination of Bcl-2 expression and the provision of a culture environment that mimicked that of the GC [CD40 ligation and interleukin 4 (IL-4)] could increase the ability of B lymphoma cells to resist drug-induced apoptosis. A Burkitt lymphoma (BL) cell line transfected with either human bcl-2 (BL-bcl-2) or control plasmid (BL-Sv2) was used to examine the effects of Bcl-2 overexpression on the cellular response and long-term survival after treatment with the DNA-alkylating drug chlorambucil (CMB) in the presence or absence of CD40 ligation and IL-4. Administration of 20 microM CMB completely prevented cell proliferation. This was associated with an increase in p53 protein levels within 24 h, without an elevation in p21, Bax, or Mdm2 proteins. Analyses of cell cycle distribution and of cyclin B expression demonstrated that both cell lines arrested at G2/M, where they died. Fifty % of BL-Sv2 cells died within 2 days, whereas 50% cell death was not observed in the BL-bcl-2 cultures until 6 days had passed. Cross-linking of CD40 with a monoclonal antibody elevated Bcl-xL protein levels by 3 h and also provided a delay in CMB-induced death. Ninety-six h after the addition of 20 microM CMB, 78% of the BL-Sv2 cells were apoptotic, whereas ligation of CD40 on BL-Sv2 cells reduced the proportion of apoptotic cells to 38%. Overexpression of Bcl-2 (in BL-bcl-2 cells) reduced apoptosis to 41%. However, when the BL-bcl-2 cells were treated with CMB together with ligation of CD40, apoptosis was reduced further to only 17% at 96 h. The Bcl-2-mediated delay in the execution of CMB-induced apoptosis did not translate significantly to increased clonogenicity. In contrast, the provision of BL-Sv2 cells with an ability to interact with the adhesion molecule vascular cell adhesion molecule-1, CD40 ligation, and IL-4 significantly increased clonogenic survival, and this was improved in BL-bcl-2 cells exposed to these GC-derived signals. These data demonstrate that the kinetics of drug-induced apoptosis can be modulated by Bcl-2 as well as by IL-4, vascular cell adhesion molecule-1, and CD40 ligation, the latter possibly involving the function of Bcl-xL. That these factors appear to act together to enhance proliferative potential after DNA damage has important implications regarding the development of drug resistance in B-cell lymphomas and future strategies for improved chemotherapy.
Bcl-2在体外可抑制药物诱导的细胞凋亡，不过在很多情况下，这仅导致细胞死亡延迟发生。细胞外环境中的体内生存信号也可能导致耐药，并可能与Bcl-2共同作用以促进细胞长期存活。生发中心（GC）内B淋巴细胞上CD40的连接可在体外抑制钙离子载体或抗IgM诱导的细胞凋亡。我们探究了Bcl-2表达与提供模拟GC的培养环境（CD40连接和白细胞介素4（IL-4））相结合是否能增强B淋巴瘤细胞抵抗药物诱导凋亡的能力。用转染了人bcl-2（BL-bcl-2）或对照质粒（BL-Sv2）的伯基特淋巴瘤（BL）细胞系，来检测在存在或不存在CD40连接和IL-4的情况下，Bcl-2过表达对经DNA烷化剂苯丁酸氮芥（CMB）处理后的细胞反应和长期存活的影响。给予20μM CMB可完全阻止细胞增殖。这与24小时内p53蛋白水平升高有关，而p21、Bax或Mdm2蛋白水平未升高。细胞周期分布和细胞周期蛋白B表达分析表明，两种细胞系均停滞在G2/M期并在该阶段死亡。50%的BL-Sv2细胞在2天内死亡，而在BL-bcl-2培养物中直到6天后才观察到50%的细胞死亡。用单克隆抗体交联CD40可使Bcl-xL蛋白水平在3小时内升高，也可延迟CMB诱导的死亡。加入20μM CMB后96小时，78%的BL-Sv2细胞发生凋亡，而BL-Sv2细胞上CD40的连接使凋亡细胞比例降至38%。Bcl-2过表达（在BL-bcl-2细胞中）使凋亡率降至41%。然而，当BL-bcl-2细胞用CMB处理并同时进行CD40连接时，96小时时凋亡率进一步降至仅17%。Bcl-2介导的CMB诱导凋亡执行延迟并未显著转化为克隆形成能力增加。相反，赋予BL-Sv2细胞与黏附分子血管细胞黏附分子-1相互作用的能力、CD40连接和IL-4可显著增加克隆形成存活率，在暴露于这些GC衍生信号的BL-bcl-2细胞中这种情况有所改善。这些数据表明，药物诱导凋亡的动力学可被Bcl-2以及IL-4、血管细胞黏附分子-1和CD40连接所调节，后者可能涉及Bcl-xL的功能。这些因素似乎共同作用以增强DNA损伤后的增殖潜力，这对于B细胞淋巴瘤耐药性的发展以及改进化疗的未来策略具有重要意义。
