Direct evidence for the participation of bcl-2 in the regulation by retinoic acid of the Ara-C sensitivity of leukemic stem cells.
Direct evidence for the participation of bcl-2 in the regulation by retinoic acid of the Ara-C sensitivity of leukemic stem cells.
复制标题
bcl-2 参与视黄酸调节白血病干细胞 Ara-C 敏感性的直接证据。
作者：
Z.;M. Minden;E. McCulloch
全反式维甲酸（ATRA）增加AML母细胞对阿拉伯糖胞嘧啶（Ara-C）或柔红霉素（DNR）的敏感性。我们已经提出bcl-2的下调是ATRA调节药物敏感性的机制的一部分。为了验证这一假设，我们将编码bcl-2的cDNA转染到连续细胞系OCI/AML-2和OCI/AML-5细胞中。分离到4个转染系；其中3个含有正义取向的bcl-2(AML5-BCL2sa、AML5-BCL2sb和2-bcl2)， 1个含有反义取向的bcl-2（AML5-bcl2as）。Northern blot证实转染基因的存在；Western blotting证实转染的基因可翻译成蛋白质。感觉定向转染bcl-2的系对Ara-C和H2O2的敏感性显著低于亲本系；转染反义基因的细胞比亲本更敏感，但差异无统计学意义。比较ATRA对转染细胞及其亲本中bcl-2表达的影响；Northern blotting结果显示，ATRA暴露后，内源基因下调，而转染基因不下调。通过获得ATRA处理细胞的Ara-C存活曲线，检测转染基因的细胞对ATRA的应答能力。与未暴露于ATRA的对照组相比，转染细胞在ATRA治疗后对Ara-C敏感性的变化很小或统计学上不显著。我们得出结论，从转染的数据提供了证据，bcl-2的表达是对Ara-C和H2O2敏感性的决定因素；ATRA对敏感性的影响需要与调控元件相关的bcl-2基因存在。
All-trans retinoic acid (ATRA) increases the sensitivity of AML blast cells to cytosine arabinoside (Ara-C) or daunorubicin (DNR) when ATRA is given after drug. We have proposed that down-regulation of bcl-2 is part of the mechanism by which ATRA regulates drug sensitivity. To test this hypothesis cDNA encoding bcl-2 was transfected into cells of the continuous lines OCI/AML-2 and OCI/AML-5. Four transfectant lines were isolated; three contained transfected bcl-2 in the sense orientation (AML5-BCL2sa, AML5-BCL2sb and 2-bcl2) and one with anti-sense bcl-2(AML5-bcl2as). The presence of the transfected gene was demonstrated by Northern blot; translation of the sense transfected genes into protein was demonstrated by Western blotting. Lines with sense-oriented transfected bcl-2 were significantly less sensitive to Ara-C or H2O2 than the parental lines; the cells with anti-sense transfected genes were more sensitive than their parent but the difference did not reach statistical significance. The effect of ATRA on bcl-2 expression was compared in sense-transfected cells and their parents; by Northern blotting it was shown that the endogenous but not the transfected genes were down-regulated after ATRA exposure. The capacity of cells with transfected genes to respond to ATRA was tested by obtaining Ara-C survival curves for ATRA-treated cells. Compared to controls not exposed to ATRA, the transfected cells showed little or statistically insignificant changes in Ara-C sensitivity after ATRA treatment. We conclude that data from the transfectants provides evidence that expression of bcl-2 is a determinant of sensitivity to Ara-C and H2O2; and that the effect of ATRA on sensitivity requires the presence of bcl-2 genes in association with regulatory elements.