Previous studies have suggested that treatment of Gleevec could inhibit TA through suppressing hTERT
mRNA level and hTERT phosphorylation level; the latter is regulated by serine/threonine protein kinase AKT [20
]. However, the mechanism by which Gleevec inhibits TA in BCR-ABL positive cells remains largely unknown. Given the clinical significance of BCR-ABL in leukemia treatment, we sought to investigate the roles of BCR-ABL in CML and its relationship with telomerase regulation, in order to facilitate in the development of better anti-CML drugs. We found that Gleevec inhibits TA through BCR-ABL by two separate mechanisms: (1) by reducing the hTERT
mRNA level via suppressing BCR-ABL mediated STAT5 signaling pathway; (2) by inhibiting phosphorylation of hTERT that can reduce TA and induce hTERT cellular translocation.
Our RT-PCR results showed that hTERT
mRNA is dramatically reduced in the presence of Gleevec. The reduction of expression is only found in hTERT
but not in hTER
of telomerase in K562 cells. This implies that Gleevec only affects the catalytic component of telomerase. Moreover, Gleevec treatment of K562 cells resulted in a significant decrease in TA but has no effect on the processivity of the telomerase. Our results are consistent with previous findings that TA is inhibited in BCR-ABL-positive cells by Gleevec and this inhibition is specific to telomerase [25
It is known that telomerase inhibition can reduce telomere length to a critical threshold resulting in senescence and/or apoptosis. We examined Gleevec's effect on telomere length in K562 cells and observed telomere shortening following 3 weeks of exposure to sub-apoptotic concentrations of Gleevec, while short-term Gleevec treatment showed no significant effect on telomere length. The efficacy of long-term telomerase inhibition suggests that Gleevec may inhibit K562 cell growth and proliferation by modulating telomere length.
From the microarray analysis, we found that PI3K
was downregulated in the JAK/STAT signaling pathway in Gleevec-treated K562 cells as compared to the K562 control group. Previous study has shown that BCR-ABL activates PI3Ks and extracellular signals to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3
), which is a second messenger that activates and recruits downstream effector proteins such as the serine/threonine kinase AKT [38
]. Thus, this suggests that the downregulation of PI3K is due to the inhibition of BCR-ABL tyrosine kinase activity via the JAK/STAT pathway upon Gleevec treatment in K562 cells, which may ultimately reduce TA in these cells.
STAT family proteins function as downstream effectors of a variety of cytokines and growth factors. STAT factors transmit signals to the nucleus where they bind to specific DNA promoter sequences and thereby regulate gene expression [39
]. Numerous studies have demonstrated that constitutively activated STAT factors, particularly STAT3 and STAT5, have been found in a wide variety of human tumors, including blood malignancies (leukemias, lymphomas) [40
]. Constitutively activated STAT factors are linked to persistent activity of tyrosine kinases, such as BCR-ABL, Src, and many others.
In this study, we observed and confirmed that STAT5 was phosphorylated only in BCR-ABL positive K562 cells and 2-hour Gleevec treatment completely abolished the phosphorylation of STAT5, which is in agreement with previous findings describing that STAT5 pathway is constitutively activated by p210 BCR-ABL and p190 BCR-ABL in leukemic cells [42
]. BCR-ABL directly phosphorylates STAT5 at tyrosine residues and promotes dimerization of phosphorylated STAT5 followed by nuclear translocation of the dimers that then promote activation of downstream target genes, which are important to induce or maintain cancer cell growth and survival. A previous report showed that inhibition of BCR-ABL, as well as STAT5, by a selective inhibitor, suppressed cell proliferation and induced apoptosis in the BCR-ABL/STAT5 double positive K562 CML cell line, while this inhibitor had no effect on either a BCR-ABL-negative/STAT5-positive or a BCR-ABL/STAT5 double-negative myeloid cell line, suggesting that the STAT5 signaling pathway leading to growth and survival is BCR-ABL-dependent [44
]. Some studies demonstrated that STAT5 activation is absolutely essential for leukemic cells because STAT5 activation leads to increased expression of genes driving cell cycle progression and promoting survival [45
], but it still remains unclear whether STAT5 is involved in regulating telomerase, which plays critical role in tumor cell growth and proliferation.
We present here several lines of evidence for a role of STAT5 in telomerase regulation in BCR-ABL positive K562 cells. We have shown that Gleevec treatment reduced STAT5 phosphorylation, which coincides with a decrease in hTERT
mRNA expression. We also found that STAT5 inhibitor selectively suppressed hTERT
mRNA expression and TA in BCR-ABL positive K562 cells. It has been known that STAT5 comprises of two highly homologous genes encoding STAT5a and STAT5b [47
]. Although these two STAT proteins share considerable functional overlap, gene-disruption experiments have revealed that STAT5a and STAT5b are functionally not redundant [48
]. Previous studies demonstrated that STAT5a mediates prolactin signaling along with mammary gland development [48
], whereas knockdown of STAT5b abrogates sexually dimorphic liver gene regulation and is associated with loss of male characteristic body growth rates [51
]. In this study, we demonstrated that STAT5a, but not STAT5b, expression and phosphorylation correlated with hTERT
gene expression and TA. More importantly, knockdown of STAT5a as well as Gleevec treatment severely reduced hTERT
gene expression and TA in BCR-ABL positive K562 cells but not in BCR-ABL negative HL60 cells. These results strongly support the notion that constitutive activation of STAT5a is likely to make a significant contribution to the telomerase regulation in BCR-ABL positive CML cells, suggesting that STAT5a could be an attractive target for the treatment of CML, especially in cases of multiple inhibitor-resistant CML. Our findings are in accordance with recent reports which demonstrated that STAT5 accounts for the resistance against Gleevec and inhibition of STAT5 can effectively decrease survival of CML cells resistant to tyrosine kinase inhibitors [52
It is known that protein phosphorylation is an important post-translational regulation controlling protein structure and function [54
]. Some reports indicated that PKC can stimulate TA through phosphorylation of hTERT, while TA was markedly inhibited in the presence of protein phosphatase 2A (PP2A) [55
]. These findings suggest that PKC and PP2A are involved in reciprocally controlling TA through phosphorylation and dephosphorylation. In addition to PKC, AKT was also found to phosphorylate the serine residue at position 824 of hTERT and stimulate TA [20
]. In our study, immunoprecipitation assay demonstrated that the hTERT tyrosine phosphorylation level was higher in K562 cells compared to HL60 cells and Gleevec treatment could effectively abrogate hTERT tyrosine phosphorylation in K562 cells as well as TA inhibition, suggesting that BCR-ABL could also phosphorylate hTERT and this phosphorylation may be important for TA maintenance and regulation. However, further investigations are necessary to determine which tyrosine could be the substrate of BCR-ABL. Previous results demonstrated that c-ABL, a non-receptor tyrosine kinase, can directly interact with hTERT and inhibit TA following phosphorylation of hTERT [56
]. This suggests that c-ABL plays a negative role in regulating telomerase function and as such we determined whether c-ABL could affect TA and hTERT protein level in c-ABL-/-
mouse embryonic fibroblasts (MEFs). We found that there was no significant effect on TA and hTERT expression by the c-ABL deficiency (Additional file 1
: Figure S13).
Previously, Liu and colleagues reported that phosphorylation of hTERT may be an important mechanism to regulate hTERT subcellular translocation from the cytosol to the nucleus [37
]. Presumably, the translocation of hTERT from a non-functional cytosolic location to a physiologically relevant nuclear location may play an important role in regulating TA in cells. As we revealed here that hTERT could be phosphorylated by BCR-ABL, we next questioned whether BCR-ABL could also govern hTERT translocation in different cellular compartments. Our confocal images have shown that hTERT in K562 BCR-ABL positive cells were localized and concentrated in nucleoli at normal conditions. Under Gleevec treatment, most of the hTERT dissociated from nucleoli into the nucleoplasm. In contrast, this phenomenon was not observed in HL60 and Jurkat, BCR-ABL deficient cells. This implies that Gleevec treatment could possibly inhibit phosphorylation of hTERT, induce hTERT translocation and thereby decrease telomerase enzyme assembly and subsequent activity.
We suppose that hTERT could be phosphorylated by BCR-ABL directly since hTERT tyrosine phosphorylation level was found elevated in K562 cells by immunoprecipitation assay (Figure ). In addition, the expression level of hTERT was similar in both cells. This result suggests that hTERT could be phosphorylated by BCR-ABL. Moreover, as shown in Figure , Gleevec treatment resulted in near-elimination of hTERT phosphorylation at tyrosine residues compared to control. We also demonstrated that the decrease in tyrosine phosphorylation of hTERT was not due to reduced hTERT expression level (Figure ). However, our immunoprecipitation results showed that neither c-ABL nor BCR-ABL interacts with hTERT directly, which contradicts to a previous study that reported the association of c-ABL with hTERT [56
]. This may due to the low affinity binding of BCR-ABL to hTERT or their transient interaction. A previous study has shown that BCR-ABL is a large protein mainly found in the cytoplasm, whereas hTERT is mostly localized in the nucleus, excluding the possibility of a direct association between the two proteins [25
]. This also implies that there may be another indirect regulation of BCR-ABL on hTERT, which may require alternative pathways or through some other intermediate proteins.
Taken together, we show here that BCR-ABL inhibition by Gleevec treatment has a significant impact on telomerase regulation based on our findings (Figure ). Our study reveals a link between transcription factor STAT5a and hTERT gene expression in BCR-ABL positive CML cell lines. Inhibition of BCR-ABL, and thus STAT5a, by Gleevec leads to reduced TA and hTERT mRNA expression as well as downregulation of hTERT phosphorylation at tyrosine residues at the post-translational level. In addition to that, we also found that BCR-ABL might regulate TA through the Wnt signaling pathway (unpublished data). These findings support the notion that telomerase expression and activity could be regulated at multiple levels by the same protein. Shuttling of telomerase in and out of nucleoli induced by Gleevec treatment provides a new insight on BCR-ABL regulated TA.
Hypothetical model of BCR-ABL tyrosine kinase regulation on TA in K562 cells.
The introduction of Gleevec has revolutionized the treatment of CML. Despite significant hematologic and cytogenetic responses, there has been concern over the emergence of resistance to Gleevec, which is mostly due to point mutations in the BCR-ABL kinase domain. One such mutation, T315I, renders CML cells completely resistant not only to Gleevec but also to second generation BCR-ABL inhibitors nilotinib and dasatinib [57
]. This has spurred the interest in developing novel tyrosine kinase inhibitors or treatment strategies to overcome the mechanisms of resistance that have led to treatment failures. Our findings showed that STAT5, more particularly, STAT5a, plays a critical role in TA regulation, suggesting that inhibition of STAT5a in combination with BCR-ABL may provide an alternative approach for treatment of leukaemia, especially in patients who are resistant to tyrosine inhibitors. Knockdown and inhibition of constitutively active STAT5 has been implicated in growth suppression in CML cells but not in normal cells [58
]. Such a combination may allow less dose of each drug, and therefore decrease side effects. More importantly, this strategy can decrease the emergence of drug-resistant cells.