Abstract
The role of p27kip1 in Chronic Myeloid Leukemia (CML) has been well studied in relation to its function as a cell cycle inhibitor. However, its cytoplasmic function especially in CML remains to be seen. We studied the localization of p27kip1 and its function during the progression of CML from chronic to blast phase. Our investigations revealed an increased localization of p27kip1 in the cytoplasm of CD34+ cells in the blast phase compared to chronic phase. Cytoplasmic p27kip1 was found to modulate RhoA activity in CD34+ stem and progenitor cells. Further, RhoA activity was shown to be dependent on cytoplasmic p27kip1 which in turn was dependent on p210Bcr-Abl kinase activity. Interestingly, RhoA activity was observed to affect cell survival in the presence of imatinib through the SAPK/JNK pathway. Accordingly, inhibition of SAPK/JNK pathway using SP600125 increased apoptosis of K562 cells in presence of imatinib. Our results, for the first time, thus reveal a crucial link between cytoplasmic p27kip1, RhoA activity and SAPK/JNK signalling. To this effect we observed a correlation between increased cytoplasmic p27kip1, increased RhoA protein levels, decreased RhoA-GTP levels and increased SAPK/JNK phosphorylation in blast phase CD34+ cells compared to chronic phase CD34+ cells.
Highlights
Chronic Myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the presence of p210Bcr-Abl fusion protein with a constitutively active tyrosine kinase activity [1]
Cytoplasmic localization of p27kip1 is dependent on p210Bcr-Abl activity
K562 cells which express p210Bcr-Abl were subjected to sub cellular fractionation and p27kip1 levels in nuclear and cytoplasmic fractions were determined. p27kip1 was found to be mostly cytoplasmic (Supplementary Information S3A). When these cells were treated with imatinib followed by immuno-cytochemistry and confocal microscopy based imaging study, p27kip1 was found to localise both in the nucleus as well as in the cytoplasm of the cells (Supplementary Information S3B)
Summary
Chronic Myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the presence of p210Bcr-Abl fusion protein with a constitutively active tyrosine kinase activity [1]. P210Bcr-Abl has been shown to promote cell cycle progression by down regulating the expression of p27kip1 [10]. P210Bcr-Abl induces the expression of Skp and promotes the degradation of p27kip1 [11,12] Another mode of regulation involves p210Bcr-Abl induced mislocalization of p27kip. Another mode of regulation involves p210Bcr-Abl induced mislocalization of p27kip1 All these processes enable p210Bcr-Abl to control cell cycle progression [13,14]. We attempted to understand the importance of cytoplasmic localization of p27kip and its impact on the progression of CML from an initial chronic phase to advanced blast phase. Cytoplasmic p27kip interacts with RhoA and thereby regulates the activity of RhoA protein These interactions are further guided by p210BcrAbl and inhibition of p210Bcr-Abl leads to changes in cytoplasmic localization of p27kip as well as RhoA activity. We present evidence that inhibition of RhoA signaling and SAPK/JNK pathway promotes cell death of K562 cells in presence of imatinib
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