Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors

Dorsey, Jay F.; Cunnick, Joan E.; Lanehart, Rheta E.; Huang, Mei; Kraker, Alan J.; Bhalla, Kapil N.; Jove, Richard; Wu, Jie

doi:10.1038/sj.leu.2402678

Cited by 42 publications

(46 citation statements)

References 25 publications

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“…[27][28][29][30] We observed that blocking JAK2 or MEK1/2 abolished the protective effect of co-culture on imatinib-induced apoptosis. However, only JAK2 inhibition was able to induce apoptosis in imatinib-or dasatinib-resistant cells.…”

Section: Discussionmentioning

confidence: 66%

BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism

et al. 2008

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Patients with chronic myeloid leukemia who become resistant to the Abl kinase inhibitor imatinib can be treated with dasatinib. This sequential treatment can lead to BCR-ABL mutations conferring broad resistance to kinase inhibitors. To model the evolution of resistance, we exposed the mouse DA1-3b BCR-ABL þ leukemic cell line to imatinib for several months, and obtained resistant cells carrying the E255K mutation. We then exposed these cells to dasatinib, and obtained dasatinib-resistant cells with composite E255K þ T315I mutations. Subcloning isolated a minor clone also carrying V299L. In co-culture, mutated cells were able to spread resistance to non-mutated cells through overexpression of interleukin 3, activation of MEK/ERK and JAK2/STAT5 pathways, and downregulation of Bim. Even the presence of less than 10% of mutated cells was sufficient to protect non-mutated cells. Blocking JAK2 and MEK1/2 inhibited the protective effect of co-culture. Mutated cells were also sensitive to JAK2 inhibition, but blocking MEK1/2 alone, or in association with kinase inhibitors, had little effect. These data indicate that sequential Abl kinase inhibitor therapy can generate sub-populations of mutated cells, which may coexist with non-mutated cells and protect them through a paracrine mechanism. Targeting JAK2 could eliminate both populations.

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Section: Discussionmentioning

confidence: 66%

BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism

et al. 2008

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“…However, there was no tyrosine phosphorylation of STAT1, STAT3 and STAT5 in BCR-stimulated Lyn-null DT40 cells. To corroborate these findings, we next used the Src-specific inhibitor PD173952 and performed a dose-inhibition study on wild-type DT40 cells (Dorsey et al, 2002). As shown in Figure 3a-d, the IC 50 for PD173952 for BCR-activated Lyn in DT40 cells was B1 mM.…”

Section: Resultsmentioning

confidence: 74%

Engagement of the B-cell antigen receptor activates STAT through Lyn in a Jak-independent pathway

2006

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Engagement of the B-cell antigen receptor (BCR) initiated by the Src kinase Lyn triggers rapid signaling cascades, leading to proliferation, differentiation or growth arrest of B cells. The Janus kinase (JAK)-STAT (signal transducer and activator of transcription) pathway, activated through cytokine receptors, mediates similar responses. Hypothesizing that Src and JAK pathways engage in crosstalk in B-cell signaling, we studied wild-type and Lyn-null B-cell lines, which express BCR. We found that activated BCR results in tyrosine phosphorylation of JAK-STAT, which required Lyn. To confirm that STAT activation is not due to JAK, we cloned the chicken homologs of JAK1 and JAK2 and made their antisense constructs. In cells expressing antisense JAK1 and JAK2, tyrosine phosphorylation of STAT was not inhibited following BCR stimulation. Using activation loop-specific phosphotyrosine antibodies, we did not detect phospho-JAK1 and phospho-JAK2 after BCR stimulation. The JAK inhibitor AG490 did not inhibit the tyrosine phosphorylation of Lyn or STAT after BCR simulation. An in vitro phosphorylation assay showed that Lyn directly phosphorylates STAT3. In an electrophoretic mobility shift assay, BCR stimulation led to enhanced DNA binding of the STAT3 in DT40, but not in the Lynnull cells. We conclude that BCR engagement activates the STAT pathway via Lyn, independent of JAK.

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“…While there is evidence that FLICA binds to proteins of molecular weight [18][19][20][21][22][23], which is in concordance with the molecular weight of large subunits of caspases (4 -6), there are certain puzzling observations that cannot be easily explained by the initially proposed mechanism of the binding, i.e., through interactions with the caspase active center only. Thus, if caspase active centers were the only binding sites for these reagents, one would expect a significant level of protection of these sites by the unlabeled caspase inhibitors (z-VAD-FMK, z-DEVD-FMK) via competitive binding.…”

Section: Discussionmentioning

confidence: 87%

“…These reagents were designed as affinity ligands to react covalently with the reactive enzymatic center of activated caspases. During the past two years several articles have been published (19,(21)(22)(23), including the papers authored by us (18,24,25), in which these reagents have been used to probe for caspase activation. Based on a similar principle, other probes were developed to detect activation of intracellular serine proteases (26).…”

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confidence: 99%

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

et al. 2003

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BackgroundFluorochrome‐labeled inhibitors of caspases (FLICA, e.g., FAM‐VAD‐FMK, FITC‐VAD‐FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes.MethodsApoptosis of HL‐60, Jurkat, MCF‐7 and T‐24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H2O2). Lymphocytes were induced by their mitogenic activation. Using multiparameter laser scanning and flow cytometry analysis, the correlation between FLICA binding and the number of known apoptotic indicators was examined. These included: collapse of the mitochondrial transmembrane potential; activation of caspase‐3 (detected immunocytochemically); binding of annexin V; chromatin condensation; the presence of DNA strand breaks; and loss of plasma membrane capability to exclude propidium iodide (PI). FLICA binding specificity was tested by pretreatment with z‐VAD‐FMK or z‐DEVD‐FMK.ResultsFLICA binding was subsequent to the collapse of mitochondrial transmembrane potential, nearly concurrent with caspase‐3 activation, and preceded annexin V binding, chromatin condensation, DNA fragmentation and loss of plasma membrane integrity. The predominant portion of FAM‐VAD‐FMK, FITC‐VAD‐FMK or FAM‐DEVD‐FMK binding to apoptotic cells could not be inhibited by z‐VAD‐FMK or z‐DEVD‐FMK, respectively, when the unlabeled inhibitors were added post‐induction of apoptosis.ConclusionsFLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases. Cytometry Part A 55A:50–60, 2003. © 2003 Wiley‐Liss, Inc.

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Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors

Cited by 42 publications

References 25 publications

BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism

BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism

Engagement of the B-cell antigen receptor activates STAT through Lyn in a Jak-independent pathway

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

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