Increased Cytoplasmic Localization of p27kip1 and Its Modulation of RhoA Activity during Progression of Chronic Myeloid Leukemia

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.


K562 K562 + Imatinib
(C) Efficacy of imatinib treatment as demonstrated by phosphorylation of known targets (STA3, STAT5) and pan-tyrosine phsophorylation. Pan-threonine phosphorylation was used as negative control. Ponceau S staining of the membrane blot has been used to indicate loading.
(D) mRNA expression of Skp2 normalised against HPRT1 in the CD34 + stem and progenitor cells of chronic and blast phases of CML.
(E) RhoA was immuno-precipitated from K562 cells followed by detection of p27 kip1 in the resulting immuno-precipitate by western blotting.
(F) Distribution of RhoA in the membrane and cytosolic fractions of CD34 + stem and progenitor cells of chronic and blast phase CML.
(G) Ba/F3 cells were electroporated with p210 Bcr-Abl WT and p210 Bcr-Abl T315I. The cells were fixed, permeabilized and stained with rhodamine tagged phalloidin. Expression of either of the p210 Bcr-Abl constructs caused an increase in filamentous actin content within the cells as observed by flow cytometric analysis. The decreased phosphorylated LIMK1/LIMK2 in the presence of p210 Bcr-Abl or p210 Bcr-Abl T315I was also observed by western blotting. Beta actin was used as a loading control. However, no discernable change in cell shape or size was evident.
i) Ba/F3 cell size and shape remains unaltered upon transfection with p210 Bcr-Abl or p210 Bcr-Abl T315I ii) BaF3 cells transfected with p210 Bcr-Abl or p210 Bcr-Abl T315I show increase in filamentous actin content as observed by increased staining with rhodamine-phalloidin.
iii) Ba/F3 cells transfected with p210 Bcr-Abl or p210 Bcr-Abl T315I show decreased expression of phosphorylated LIMK1/LIMK2. Densitometric analysis of the blot has been represented below.
(H) K562 cells were treated with imatinib with the indicated dose (in µM) for 24 hr and the levels of ROCK1 and ROCK2 was assessed by western blotting. Beta actin was used as a loading control.
(I) Fibronectin adhesion assay: CD34 + cells isolated from chronic CML patient was nucleofected with the various constructs of p27 and their adherence to fibronectin matrix was assayed in triplicate. Data represents fold change in cellular adhesion to fibronectin normalised against control CD34 + cells.
(J) Transwell migration assay: CD34 + cells isolated from chronic CML patient was nucleofected with the various constructs of p27 and their migration through the 3µ pore was assayed in triplicate. Data represents percentage of transfected cells which have migrated to the lower chamber with respect to control CD34 + cells.
(K) K562 cells were transfected with GFP tagged MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with/without 1µM imatinib for 24 hr. Cells were stained with anti cytochrome c antibody (red) and observed under the confocal microscope for diffused cytochrome c staining indicating release of cytochrome c from the mitochondria. 100 cells were observed in three independent preparations and the mean ± SEM was plotted. (p<0.005) (L) K562 cells were transfected with MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with/without 1µM imatinib. Cytochrome c released from the mitochondria was estimated in the purified mitochondria free cytosolic fraction by western blot. Tim23 was used to estimate mitochondrial contamination in the cytosolic preparations (densitometric estimation given in parenthesis) while beta actin was used as a loading control. (n=3, p<0.019) (P) Densitometric analysis of the western blots (Fig. 5C) showing the change in expression of phospho-c-jun and c-jun in the presence of imatinib or C3 exozyme or both. Normalised densitometric expression values were plotted against the expression seen in untreated K562 cells. (n=3, *p<0.016, **p<0.003) (Q) K562 cells were treated with SP600125, imatinib or both. Cytochrome c released from the mitochondria was estimated in the purified mitochondria free cytosolic fraction by western blot (Fig. 5D). Tim23 was used to estimate mitochondrial contamination in the cytosolic preparations while beta actin was used as a loading control. (n=3,p<0.008)