Ku Birkenkamp

Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts

In the present study, we examined the underlying mechanism, which causes the constitutive tyrosine phosphorylation of signal transducer and activator of transcription 5 (STAT5) in acute myeloid leukemia (AML) blasts. Constitutive STAT5 phosphorylation was observed in 18 of 26 (69%) patients with AML. The constitutive STAT5 phosphorylation was caused by different mechanisms. In the majority of the investigated cases (71% (12 of 17)) constitutive STAT5 phosphorylation was associated with autophosphorylation of the type III receptor tyrosine kinase Flt3. In 47% (eight of 17) of these cases autophosphorylation of Flt3 coincided with tandem duplications of the Flt3 gene, resulting in constitutive phosphorylation of the receptor, while 24% (four of 17) of the cases demonstrated STAT5 phosphorylation and Flt3 autophosphorylation without mutations. In addition, a subset of AML cases (29% (five of 17)) had no autophosphorylation of the Flt3 receptor, but demonstrated constitutive STAT5 phosphorylation, which was partly due to autocrine growth factor production. All AML cases with high STAT5 and Flt3 phosphorylation demonstrated, in general, a lower percentage of spontaneous apoptosis, compared to AML blasts with no spontaneous STAT5 phosphorylation. Addition of the receptor tyrosine III kinase inhibitor AG1296 strongly inhibited STAT5 phosphorylation and enhanced the percentage of apoptotic cells without modulating the Bcl-xl protein levels. These data indicate that in the majority of AML cases the constitutive STAT5 phosphorylation is caused by Flt3 phosphorylation mostly due to mutations in the receptors and associated with a low degree of spontaneous apoptosis.

Constitutive NF-kappa B DNA-binding activity in AML is frequently mediated by a Ras/PI3-K/PKB-dependent pathway

In the present study, we aimed to elucidate the mechanism responsible for constitutive NF-κB DNA-binding activity in AML cells. Intervening in aberrant signaling pathway provides a rational approach for in vivo targeting of AML cells. Constitutive NF-κB DNA-binding activity was observed in 16 of 22 (73%) investigated AML cases and was, in general, associated with resistance to spontaneous apoptosis. Indeed, inhibition of NF-κB activity by the NF-κB inhibitor SN-50 peptide resulted in enhanced chemotherapy-induced apoptosis. In the majority of cases, constitutive NF-κB activity was mediated by a Ras/PI3 kinase (PI3-K)/protein kinase B (PKB)-mediated pathway. The PI3-K inhibitor Ly294002 and the Ras inhibitor L-744832 both inhibited PKB phosphorylation and NF-κB DNA-binding activity. The constitutive activation of Ras GTP-ase was caused by mutations in the gene encoding for N-Ras in 29% of the cases. The constitutive NF-κB activity could so far not be ascribed to the autocrine production of growth factors or to mutations in the Flt3 receptor, since anti-GM-CSF, -IL-1, -IL6, -TNFα or the tyrosine kinase inhibitor AG1296 did not affect the NF-κB DNA-binding activity. The present study demonstrates that Ras activation is an important pathway for triggering the NF-κB pathway in AML cells.

The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway.

In the present study we investigated a possible role for the p38 mitogen‐activated protein (MAP) kinase pathway in mediating nuclear factor‐kappa B (NF‐κB) transcriptional activity in the erythroleukaemic cell line TF‐1. TF‐1 cells stimulated with the phosphatase inhibitor okadaic acid (OA) demonstrated enhanced NF‐κB and GAL4p65‐regulated transcriptional activity which was associated with elevated p38 phosphorylation. However, pretreatment with the p38 MAPK specific inhibitor SB203580 (1 μM) or overexpression of kinase‐deficient mutants of MKK3 or MKK6 did not affect OA‐enhanced NF‐κB transcriptional potency, as determined in transient transfection assays. In fact, 5 and 10 μM SB203580 enhanced rather than inhibited NF‐κB‐mediated promoter activity by 2 fold, which was independent of phosphorylation of the p65 subunit. The SB203580‐mediated increase in NF‐κB transcriptional activity was associated with enhanced phosphorylation of extracellular signal‐regulated kinase (ERK)1/2 and c‐Jun N‐terminal kinase (JNK), but not p38 kinase. Overexpression of kinase‐deficient mutants belonging to the ERK1/2, JNK, and p38 pathways showed that only dominant‐negative Raf‐1 abrogated SB203580‐enhanced NF‐κB activity. This would implicate the involvement of the ERK1/2 pathway in the enhancing effects of SB203580 on NF‐κB‐mediated gene transcription. This study demonstrates that the p38 MAP kinase pathway is not involved in the OA‐induced activation of NF‐κB. SB203580 at higher concentrations activates the ERK pathway, which subsequently enhances NF‐κB transcriptional activity.

Hydrophobins line air channels in fruiting bodies of Schizophyllum commune and Agaricus bisporus

The hydrophobin SC4 was isolated from the medium of a dikaryon from Schizophyllum commune with disrupted SC3 genes. Although not glycosylated, its biophysical properties were similar to those of SC3. As the hydrophobins SC3 from S. commune and ABH1 and ABH3 from Agaricus bisporus, SC4 self-assembled at hydrophilic-hydrophobic interfaces into an SDS insoluble amphipathic film with a typical rodlet structure at its hydrophobic face, and also proved to be a powerful surfactant. Similar rodlet structures were observed in the fruiting body plectenchyma. By immunodetection SC4 could be localized lining air channels within this tissue. A similar localization was found for the ABH1 hydrophobin in fruiting bodies of A. bisporus. Probably, these hydrophobin coatings prevent collapse of air channels allowing efficient gas exchange even under wet conditions.

A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-κB, might determine the final cellular response.

An inhibitor of PI3-K differentially affects proliferation and IL-6 protein secretion in normal and leukemic myeloid cells depending on the stage of differentiation

In this study, we examined the involvement of the phosphatidylinositol 3-kinase (PI3-K) and p70S6 kinase signal transduction pathway in the interleukin-1(IL-1)-mediated proliferation and cytokine production by normal and leukemic myeloid cells. Total AML blast populations, early progenitor (CD34(+)/CD36(-)) cells, and more differentiated (CD34(-)/CD36(+)) cells were treated with the PI3-K inhibitor Ly294002 and p70S6K inhibitor rapamycin. The effects on proliferation, IL-6 protein secretion, and intracellular signaling cascades were determined and compared with normal CD34(+) cells and monocytes. The function of the PI3-K pathway was dependent on the differentiation state of the AML cell population. In immature blasts, the IL-1-induced proliferation was strongly inhibited by Ly294002 and rapamycin, without a distinct effect on IL-6 protein production. In contrast, in mature monocytic blast cells inhibition of the PI3-K signaling route had a stimulatory effect on IL-6 protein secretion. Interestingly, these findings were not specifically linked to the malignant counterpart but were also observed with normal CD34(+) sorted cells vs mature monocytes. Evidence is provided that the Ly294002-induced increase in IL-6 protein secretion is linked to the cAMP dependent signaling pathway and not to changes in the phosphorylation of ERK or p38. However, although the enhanced IL-6 protein secretion is cAMP dependent, it was not found to be mediated by protein kinase A (PKA) or by the GTP-ase Rap1. This study indicates that inhibition of the PI3-K signaling pathway has an inhibitory effect on cell proliferation but a stimulatory effect on IL-6 expression mediated by a cAMP-dependent but PKA-independent route.