Pseudonegative BCL2 protein expression in a t(14;18) translocation positive lymphoma cell line: a need for an alternative BCL2 antibody
Pseudonegative BCL2 protein expression in a t(14;18) translocation positive lymphoma cell line: a need for an alternative BCL2 antibody
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DOI：
10.3109/00313021003631296
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发表时间：
2010-04-01
期刊：
影响因子：
4.5
通讯作者：
Mason, David Y.
中科院分区：
文献类型：
--
作者：
Masir, Noraidah;Campbell, Lisa J.;Mason, David Y.
研究方法：通过Western印迹和使用两种不同抗体的免疫组织化学分析t（14;18）阳性细胞系FL 18、Karpas-422、SU-DHL-4和SU-DHL-6中的BCL 2表达。用荧光原位杂交（FISH）技术检测细胞遗传学改变，用真实的实时定量PCR技术检测BCL 2 mRNA水平。结果：在FL 18、Karpas-422、SU-DHL-4和SU-DHL-6细胞系中，BCL 2 mRNA水平与BCL 2蛋白表达水平相关。与此相反，在SU-DHL-6细胞系中使用标准抗BCL 2抗体（BCL 2/124）未检测到BCL 2蛋白，尽管存在t（14;18）易位和高水平的mRNA。易位BCL 2的cDNA测序显示SU-DHL-6细胞系中有三个突变，其中一个导致标准BCL 2抗体识别区域的氨基酸取代（I48 F），而另外两个是aa 71和aa 72的沉默突变。有趣的是，当用替代抗体E17测试BCL 2表达时，在SU-DHL-6中检测到该蛋白质，表明使用标准抗体的SU-DHL-6系对BCL 2的“阴性”是假的。Karpas-422（G47 D，P59 L）和SU-DHL-4（P59 T，S117 R）的氨基酸发生了变化，但这些变化并不影响BCL 2的检测。结论：BCL 2基因易位后的体细胞突变可能会阻碍BCL 2抗体识别表位，从而导致标准抗体的假阴性表达。建议在实践中，所有BCL 2阴性病例应常规使用替代抗体染色，以防止假阴性。
Methods: BCL2 expression in the t(14;18) positive cell lines FL18, Karpas-422, SU-DHL-4 and SU-DHL-6, was analysed by Western blotting and by immunohistochemistry using two different antibodies. FISH analysis was performed to confirm the cytogenetic changes in the cell lines and real time quantitative PCR was used to evaluate the BCL2 mRNA level. Sequence analysis of translocated BCL2 was performed on FL18, Karpas-422, SU-DHL-4 and SU-DHL-6 cell lines.Results: In FL18, Karpas-422, and SU-DHL-4, the BCL2 mRNA level correlated with the BCL2 protein expression. In contrast, BCL2 protein was not detected in SU-DHL-6 line using standard anti-BCL2 antibody (BCL2/124), despite the presence of the t(14;18) translocation and high level of mRNA. cDNA sequencing of translocated BCL2 showed three mutations in the SU-DHL-6 cell line, one of which resulted in an amino acid substitution (I48F) in the region recognised by the standard BCL2 antibody, whereas the other two were silent mutations at aa71 and aa72. Interestingly, when BCL2 expression was tested with an alternative antibody, E17, the protein was detected in SU-DHL-6, suggesting that the 'negativity' of SU-DHL-6 line for BCL2 using the standard antibody is spurious. Amino acid changes were found in Karpas-422 (G47D, P59L) and SU-DHL-4 (P59T, S117R) but these did not affect BCL2 detection.Conclusions: This study suggests that some somatic mutations of the translocated BCL2 gene may prevent epitope recognition by BCL2 antibodies, and hence cause false negative expression using the standard antibody. It is recommended that in practice all BCL2 negative cases should routinely be stained with an alternative antibody to prevent false negativity.