High pH Lysis Allows Immunoprecipitation of p210BCR-ABL in Primary Chronic Myeloid Leukaemia.

Abstract

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder, which is characterised by the presence of the Philadelphia translocation (Ph) (t (9; 22)). The translocation occurs when parts of the c-ABL gene, located on chromosome 9 fuse with the BCR gene, located on chromosome 22. BCR-ABL fusion results in the production of p210BCR-ABL, a chimeric protein with the molecular weight of 210kda. p210BCR-ABL is located in the cytoplasm and has constitutive protein tyrosine kinase activity. Numerous publications have reported that p210BCR-ABL forms complexes with multiple cytoplasmic proteins which affect several signalling pathways. However this published data exists for cell lines expressing p210BCR-ABL and not for primary CML cells. The lack of information based on primary CML cells is due to the presence of a degradative activity which is released when cells are lysed under standard conditions, destroying p210BCR-ABL rapidly and permanently. This prevents quantification or investigation of proteins co-precipitating with p210BCR-ABL in primary CML cells. The degradative activity can be assayed by co-lysing K562 cells, a Ph+ cell line that does not contain the activity, with primary CML cells. Using this assay we show that the degradative activity found in the primary CML cells destroys the p210BCR-ABL present in the K562 cells. The same assay was used to show that the activity is present in both primary CML and normal mononuclear cells (MNC). Conventional protease inhibitor cocktails have not proven effective in inhibiting this degradative activity. We show here that the degradative activity in primary cells is associated with the cell lysosomes and may be an acid dependent hydrolase. By lysing primary CML cells at pH12 we have shown that the degradative activity can be significantly inhibited thereby allowing us to detect p210BCR-ABL in primary CML cells by western blotting. Furthermore, p210BCR-ABL can be immunoprecipitated from primary CML cells when cells are lysed at high pH; however immunoprecipitation is more effective when the pH of lysate is reduced to pH8. We demonstrate that the alterations in pH during the immunoprecipitation procedure do not disrupt the co-immunoprecipitation of p210BCR-ABL-Cbl and Crkl - Cbl associations. Thus, this method is capable of confirming or refuting in primary cells the molecular associations that have been demonstrated in CML cell lines.