MEIS1-mediated transactivation of synaptotagmin-like 1 promotes CXCL12/CXCR4 signaling and leukemogenesis.

The TALE-class homeoprotein MEIS1 specifically collaborates with HOXA9 to drive myeloid leukemogenesis. Although MEIS1 alone has only a moderate effect on cell proliferation in vitro, it is essential for the development of HOXA9-induced leukemia in vivo. Here, using murine models of leukemogenesis, we have shown that MEIS1 promotes leukemic cell homing and engraftment in bone marrow and enhances cell-cell interactions and cytokine-mediated cell migration. We analyzed global DNA binding of MEIS1 in leukemic cells as well as gene expression alterations in MEIS1-deficent cells and identified synaptotagmin-like 1 (Sytl1, also known as Slp1) as the MEIS1 target gene that cooperates with Hoxa9 in leukemogenesis. Replacement of SYTL1 in MEIS1-deficent cells restored both cell migration and engraftment. Further analysis revealed that SYTL1 promotes cell migration via activation of the CXCL12/CXCR4 axis, as SYTL1 determines intracellular trafficking of CXCR4. Together, our results reveal that MEIS1, through induction of SYTL1, promotes leukemogenesis and supports leukemic cell homing and engraftment, facilitating interactions between leukemic cells and bone marrow stroma.

Homeodomain transcription factor Meis1 is a critical regulator of adult bone marrow hematopoiesis.

Hematopoietic stem cells in the bone marrow have the capacity to both self-renew and to generate all cells of the hematopoietic system. The balance of these two activities is controlled by hematopoietic stem cell-intrinsic regulatory mechanisms as well as extrinsic signals from the microenvironment. Here we demonstrate that Meis1, a TALE family homeodomain transcription factor involved in numerous embryonic developmental processes, is selectively expressed in hematopoietic stem/progenitor cells. Conditional Meis1 knockout in adult hematopoietic cells resulted in a significant reduction in the hematopoietic stem/progenitor cells. Suppression of hematopoiesis by Meis1 deletion appears to be caused by impaired self-renewal activity and reduced cellular quiescence of hematopoietic stem/progenitor cells in a cell autonomous manner, resulting in stem cell exhaustion and defective long-term hematopoiesis. Meis1 deficiency down-regulated a subset of Pbx1-dependent hematopoietic stem cell signature genes, suggesting a functional link between them in the maintenance of hematopoietic stem/progenitor cells. These results show the importance of Meis1 in adult hematopoiesis.

Akt and PKC are involved not only in upregulation of telomerase activity but also in cell differentiation-related function via mTORC2 in leukemia cells.

We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/ßII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.

Combined analysis of cell growth and apoptosis-regulating proteins in HPVs associated anogenital tumors.

BACKGROUND: The clinical course of human papillomavirus (HPV) associated with Bowenoid papulosis and condyloma acuminatum of anogenital tumors are still unknown. Here we evaluated molecules that are relevant to cellular proliferation and regulation of apoptosis in HPV associated anogenital tumors. METHODS: We investigated the levels of telomerase activity, and inhibitor of apoptosis proteins (IAPs) family (c-IAP1, c-IAP2, XIAP) and c-Myc mRNA expression levels in 20 specimens of Bowenoid papulosis and 36 specimens of condyloma acuminatum in anogenital areas. Overall, phosphorylated (p-) AKT, p-ribosomal protein S6 (S6) and p-4E-binding protein 1 (4EBP1) expression levels were examined by immunohistochemistry in anogenital tumors both with and without positive telomerase activity. RESULTS: Positive telomerase activity was detected in 41.7% of Bowenoid papulosis and 27.3% of condyloma acuminatum compared to normal skin (p < 0.001). In contrast, the expression levels of Bowenoid papulosis indicated that c-IAP1, c-IAP2 and XIAP mRNA were significantly upregulated compared to those in both condyloma acuminatum samples (p < 0.001, p < 0.001, p = 0.022, respectively) and normal skin (p < 0.001, p = 0.002, p = 0.034, respectively). Overall, 30% of Bowenoid papulosis with high risk HPV strongly promoted IAPs family and c-Myc but condyloma acuminatum did not significantly activate those genes. Immunohistochemically, p-Akt and p-S6 expressions were associated with positive telomerase activity but not with p-4EBP1 expression. CONCLUSION: Combined analysis of the IAPs family, c-Myc mRNA expression, telomerase activity levels and p-Akt/p-S6 expressions may provide clinically relevant molecular markers in HPV associated anogenital tumors.

An activating mutation in the CSF3R gene induces a hereditary chronic neutrophilia.

We identify an autosomal mutation in the CSF3R gene in a family with a chronic neutrophilia. This T617N mutation energetically favors dimerization of the granulocyte colony-stimulating factor (G-CSF) receptor transmembrane domain, and thus, strongly promotes constitutive activation of the receptor and hypersensitivity to G-CSF for proliferation and differentiation, which ultimately leads to chronic neutrophilia. Mutant hematopoietic stem cells yield a myeloproliferative-like disorder in xenotransplantation and syngenic mouse bone marrow engraftment assays. The survey of 12 affected individuals during three generations indicates that only one patient had a myelodysplastic syndrome. Our data thus indicate that mutations in the CSF3R gene can be responsible for hereditary neutrophilia mimicking a myeloproliferative disorder.

JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disorders.

The JAK2(V617F) mutation is frequently observed in classical myeloproliferative disorders, and disease progression is associated with a biallelic acquisition of the mutation occurring by mitotic recombination. In this study, we examined whether JAK2 activation could lead to increased homologous recombination (HR) and genetic instability. In a Ba/F3 cell line expressing the erythropoietin (EPO) receptor, mutant JAK2(V617F) and, to a lesser extent, wild-type (wt) JAK2 induced an increase in HR activity in the presence of EPO without modifying nonhomologous end-joining efficiency. Moreover, a marked augmentation in HR activity was found in CD34(+)-derived cells isolated from patients with polycythemia vera or primitive myelofibrosis compared with control samples. This increase was associated with a spontaneous RAD51 foci formation. As a result, sister chromatid exchange was 50% augmented in JAK2(V617F) Ba/F3 cells compared with JAK2wt cells. Moreover, JAK2 activation increased centrosome and ploidy abnormalities. Finally, in JAK2(V617F) Ba/F3 cells, we found a 100-fold and 10-fold increase in mutagenesis at the HPRT and Na/K ATPase loci, respectively. Together, this work highlights a new molecular mechanism for HR regulation mediated by JAK2 and more efficiently by JAK2(V617F). Our study might provide some keys to understand how a single mutation can give rise to different pathologies.

Multistep regulation of telomerase during differentiation of HL60 cells.

Using three different differentiation agents (1alpha, 25 dihydroxyvitamin D3, all-trans-retinoic acid, and Am80), down-regulation of telomerase activity was found to be a common response during the monocytic or granulocytic differentiation of human acute myeloblastic leukemia cell line 60 (HL60) cells. Rapid down-regulation of telomerase transcription occurred during early differentiation of HL60 cells prior to G(1) arrest. Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity, and the extent of the suppression that occurred while maintaining telomerase protein expression suggested the post-translational regulation of telomerase activity. Recombinant Akt dose-dependently increased telomerase activity, and telomerase was inhibited at the transcriptional and post-translational levels by LY294002, suggesting that PI-3K/Akt is one of the key signaling proteins involved in telomerase regulation. Each of the three differentiation agents caused a significant increase of signaling proteins (including Akt) at 3 days after the initiation of differentiation. Changes of acetyl-histone H4, which regulates transcription of the telomerase gene, were observed before the activation of Akt. This finding suggests that epigenetic control of telomerase transcription occurs before activation of Akt during the late stage of differentiation. These results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional and the other being post-translational.

Bowen's disease with high telomerase activity.

We describe an 81-year-old Japanese woman who had a palm-sized, erythematous plaque with a nodular lesion on the lateral abdomen. The biopsy specimens taken from the erythematous plaque and reddish nodule show that bowenoid changes were present in the epidermis and epidermis to dermis, respectively. A sentinel lymph node biopsy (SNB) was performed with blue dye and radioisotope in her right groin region and two lymph nodes were found to be occupied by many atypical cells. The erythematous plaque with nodular lesion was completely removed with a 3-cm margin under general anesthesia, and complete regional lymph node dissection was also performed. In addition, high telomerase activity was seen in the erythema plaque while using a telomeric repeat amplification protocol assay. In conclusion, some instances of Bowen's disease might have high telomerase activity in the atypical cells and can progress to Bowen's carcinoma. The SNB was regarded as a useful method to detect early lymph node metastases in this case.

STAT3 and PKC differentially regulate telomerase activity during megakaryocytic differentiation of K562 cells.

Telomerase is active in immature somatic cells, but not in differentiated cells. However, the regulation during cell differentiation is not well understood. In this study, a human chronic myelogenous leukemia cell line (K562) was induced to differentiate into megakaryocytes by TPA, and erythroid by STI571. A human acute myeloblastic leukemia cell line (HL60) was also induced to differentiate into monocytes by TPA and VD3, and granulocyte by ATRA. TPA induced transient increase of telomerase activity (mainly nuclear fraction) during megakaryocytic differentiation, while the expression of hTERT decreased gradually throughout the same period. Pretreatment with PKC inhibitors inhibited the megakaryocytic differentiation, transient increase of telomerase activity, while recombinant PKC increased telomerase activity. ChIP assay resulted STAT3 and STAT5 dissociated from the hTERT promoter, indicating that STAT3 and STAT5 are one of the transcriptional regulators. These results suggest that telomerase activity is regulated by two mechanisms during megakaryocytic differentiation.