Conventional Western blotting techniques will not reliably quantify p210^BCR-ABL1 levels in CML mononuclear cells

We read with interest the paper by Copland et al,¹  in which they used quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blotting to demonstrate that the transcript and protein levels of p210^BCR-ABL1are elevated in primitive chronic myeloid leukemia (CML) progenitors relative to more mature cells. The work adds substance to the growing body of evidence for increased p210^BCR-ABL1 activity in the putative CML stem cell, which also provides a partial rationale for the failure of treatments such as imatinib to achieve complete eradication of disease in vivo.

We note that the RT-PCR–based quantitative data and patterns of CrkL phosphorylation that Copland et al¹  report support the hypothesis that p210^BCR-ABL1 activity is enhanced in more primitive cell populations. However, their Western blotting method uses a conventional lysis buffer to lyse cells in the cold, which is standard procedure. It has been known since 1987²  that such lysis of CML mononuclear cells (MNCs) releases a degradative activity that very rapidly and selectively destroys p210^BCR-ABL1 and c-ABL but does not degrade other proteins.³  This activity has been reported to be primarily restricted to the mature cell compartments,^2-4  hence its influence is commensurately greater in MNC lysates. It is entirely predictable therefore that Copland et al¹  could not recover a p210^BCR-ABL1 signal or a 210-kDa phosphotyrosine signal from a CML MNC blot and that the signal recovered is greater from CD34⁺CD38⁻ cells than CD34⁺ cells. CD34⁺ cell fractions typically contain up to 5% contaminating mature cells that would elicit some signal degradation, while double-sorted CD34⁺CD38⁻ populations would be expected to contain fewer contaminating cells.

Furthermore, while Copland et al²  could not recover any p210^BCR-ABL1 signal from CML MNCs, Guo et al⁵  showed that use of an extremely toxic nerve agent and a boiling lysis medium permitted routine p210^BCR-ABL1recovery from CML MNCs. We have recently published data showing that the degradative activity is probably an acid-dependent hydrolase and can be neutralized by a high-pH lysis regimen to allow accurate determination of p210^BCR-ABL1protein levels. This has the further advantage of permitting coimmunoprecipitation studies, and so, for the first time, investigation of p210^BCR-ABL1-protein complexes in primary cells from CML patients is now possible.³  These complexes play an important role in CML pathogenesis, and the amount of p210^BCR-ABL1present will be a significant contributory factor. The degradation of p210^BCR-ABL1by MNC components of primary cell lysates is therefore a critical variable. Future Western blotting experiments should take account of this inhibitory activity.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Myrtle Y. Gordon, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom; e-mail: myrtle.gordon@imperial.ac.uk.