Dole M G, Jasty R, Cooper M J, Thompson C B, Nuñez G, Castle V P
Department of Pediatrics, University of Michigan, Ann Arbor 48109-0684, USA.
Cancer Res. 1995 Jun 15;55(12):2576-82.
bcl-x is a new member of the bcl-2 gene family and is highly expressed in neural tissues. The present study was designed to determine the expression of the bcl-x gene products in neuroblastoma (NB) and their role in the modulation of chemotherapy-induced apoptosis. Twenty-seven NB cell lines were screened by quantitative immunoprecipitation for Bcl-xL, Bcl-xS, and Bcl-2 expression. None of the cell lines expressed Bcl-xS. Twenty-four of 27 (88%) of the NB cell lines expressed Bcl-xL and 21 of 27 (78%) were positive for Bcl-2. The level of Bcl-xL and Bcl-2 expression was variable among the lines analyzed. Bcl-2 expression was restricted to cells of chromaffin lineage, whereas Bcl-xL was seen in both chromaffin and nonchromaffin lines. To determine whether Bcl-xL could mediate chemotherapy resistance, a NB cell line expressing negligible levels of Bcl-xL was transfected with a bcl-xL expression vector, and unique clones were generated expressing variable levels of Bcl-xL. Cells were treated either with cisplatinum (CP), 4-hydroperoxy-cyclophosphamide (4-HC), or etoposide (VP-16) to induce apoptosis, and cell viability and DNA degradation were determined. Following treatment with CP or 4-HC, Bcl-xL-expressing cells showed significantly increased viability as compared to vector-transfected controls (P < 0.005). Flow cytometric analysis of propidium iodide-stained nuclei following CP or 4-HC treatment revealed significantly increased DNA degradation in controls as compared to Bcl-xL-expressing lines (P < 0.004). DNA analysis by pulsed-field gel electrophoresis revealed high molecular weight (approximately 40 kb) DNA degradation in controls, whereas the DNA in cells expressing Bcl-xL was largely intact. In contrast to CP and 4-HC, results with VP-16 revealed a short-term delay in the onset of apoptosis in Bcl-xL-expressing cells with no long-term survival advantage. The results of these studies indicate Bcl-xL is expressed in NB cells and functions in a manner analogous to Bcl-2 by inhibiting chemotherapy-induced apoptosis.
bcl-x是bcl-2基因家族的一个新成员，在神经组织中高度表达。本研究旨在确定bcl-x基因产物在神经母细胞瘤（NB）中的表达及其在调节化疗诱导的细胞凋亡中的作用。通过定量免疫沉淀法筛选了27种NB细胞系，检测其Bcl-xL、Bcl-xS和Bcl-2的表达情况。所有细胞系均未表达Bcl-xS。27种NB细胞系中有24种（88%）表达Bcl-xL，27种中有21种（78%）Bcl-2呈阳性。在所分析的细胞系中，Bcl-xL和Bcl-2的表达水平各不相同。Bcl-2的表达仅限于嗜铬细胞系细胞，而Bcl-xL在嗜铬细胞系和非嗜铬细胞系中均可见。为了确定Bcl-xL是否能介导化疗耐药性，将一个几乎不表达Bcl-xL的NB细胞系用bcl-xL表达载体进行转染，产生了表达不同水平Bcl-xL的独特克隆。用顺铂（CP）、4-氢过氧环磷酰胺（4-HC）或依托泊苷（VP-16）处理细胞以诱导凋亡，并测定细胞活力和DNA降解情况。用CP或4-HC处理后，与载体转染对照相比，表达Bcl-xL的细胞活力显著增加（P < 0.005）。CP或4-HC处理后，对碘化丙啶染色细胞核进行流式细胞术分析显示，与表达Bcl-xL的细胞系相比，对照中的DNA降解显著增加（P < 0.004）。脉冲场凝胶电泳DNA分析显示对照中有高分子量（约40 kb）DNA降解，而表达Bcl-xL的细胞中的DNA基本完整。与CP和4-HC不同，VP-16的结果显示表达Bcl-xL的细胞凋亡起始有短期延迟，但没有长期生存优势。这些研究结果表明，Bcl-xL在NB细胞中表达，其功能类似于Bcl-2，可抑制化疗诱导的细胞凋亡。
