竞争性聚合酶链反应作为检测慢性髓性白血病bcr-abl基因扩增的一种方法。

Competitive polymerase chain reaction as a method to detect the amplification of bcr-abl gene of chronic myeloid leukemia.

作者信息

Campanini F, Santucci M A, Pattachini L, Brusa G, Piccioli M, Barbieri E, Babini L, Tura S

机构信息

Istituto di Ematologia e Oncologia Medica L.A. Seràgnoli, via Massarenti, 9, Ospedale S. Orsola, Università degli Studi di Bologna, 40138 Bologna, Italy.

出版信息

Haematologica. 2001 Feb;86(2):167-73.

PMID:11224486

Abstract

BACKGROUND AND OBJECTIVES

The chimeric product of the bcr-abl rearranged gene is critical in the pathogenesis of chronic myeloid leukemia (CML), yet its role in the progression of the disease remains unclear. There is some evidence that increased bcr-abl expression levels, possibly due to gene amplification, precede the clonal evolution of CML hematopoietic progenitors toward a fully transformed phenotype and might be involved in their resistance to interferon-alpha or tyrosine kinase inhibitors.

DESIGN AND METHODS

To quantify the bcr-abl gene both at the genomic and at the transcriptional levels we developed a competitive polymerase chain reaction (PCR) strategy. The competitive PCR technique is based upon the co-amplification of the sample template (target) together with increasing amounts of a DNA fragment (competitor) sharing with the target the primer recognition sites, but differing in size. We constructed a competitor for the quantification of both b2a2 and b3a2 alternative splicing forms of the bcr-abl chimera and established the accuracy and reproducibility of our competitive strategy in a clone of the murine 32DG hematopoietic cell line (32D LG7), which bears a stable integration of a single copy of p210 bcr-abl fusion gene. We utilized this technique to follow, over a period of 200 days, the fusion gene copy numbers and transcription rates in several p210 bcr-abl-transduced 32D cell clones, an experimental condition mimicking the evolution of CML myeloid progenitors in vivo.

RESULTS

Our results are consistent with p210 bcr-abl overexpression but not gene amplification associated with their clonal evolution. Increased p210 bcr-abl transcription rate is associated with the abrogation of radiation-induced apoptotic cell death, suggesting a role for the chimeric gene expression level in cell life expectancy after a genotoxic insult.

INTERPRETATION AND CONCLUSIONS

We conclude that the assessment of gene amplification and expression might serve to improve prognostic classification and follow-up of CML patients.

摘要

背景与目的

bcr-abl重排基因的嵌合产物在慢性髓性白血病（CML）的发病机制中至关重要，但其在疾病进展中的作用仍不清楚。有证据表明，bcr-abl表达水平升高（可能由于基因扩增）先于CML造血祖细胞向完全转化表型的克隆进化，并且可能参与其对α干扰素或酪氨酸激酶抑制剂的耐药性。

设计与方法

为了在基因组和转录水平定量bcr-abl基因，我们开发了一种竞争性聚合酶链反应（PCR）策略。竞争性PCR技术基于样品模板（靶标）与越来越多的DNA片段（竞争物）共同扩增，该DNA片段与靶标共享引物识别位点，但大小不同。我们构建了一个竞争物，用于定量bcr-abl嵌合体的b2a2和b3a2可变剪接形式，并在携带单个拷贝p210 bcr-abl融合基因稳定整合的小鼠32DG造血细胞系（32D LG7）的一个克隆中确定了我们竞争策略的准确性和可重复性。我们利用该技术在200天的时间内追踪了几个p210 bcr-abl转导的32D细胞克隆中的融合基因拷贝数和转录率，这一实验条件模拟了CML髓系祖细胞在体内的进化。