Selective cytotoxicity of withaphysalins in myeloid leukemia cell lines versus peripheral blood mononuclear cells

Life Sciences ◽  
2006 ◽  
Vol 79 (18) ◽  
pp. 1692-1701 ◽  
Author(s):  
Danilo Damasceno Rocha ◽  
Gardenia Carmen Gadelha Militão ◽  
Maria Leopoldina Veras ◽  
Otília Deusdênia Loiola Pessoa ◽  
Edilberto Rocha Silveira ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Peripheral Blood Mononuclear ◽  
Selective Cytotoxicity ◽  
Leukemia Cell Lines ◽  
Blood Mononuclear Cells ◽  
Myeloid Leukemia Cell

Hypoxia Blocks Activation of JAK2V617F Through Suppression of SHP-2 Expression in MPN Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 126-126
Author(s):  
Toru Mitsumori ◽  
Yumi Nozaki ◽  
Norio Komatsu ◽  
Keita Kirito
Keyword(s):  
Stem Cells ◽  
Polycythemia Vera ◽  
Cell Lines ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Peripheral Blood Mononuclear ◽  
Leukemia Cell Lines ◽  
Blood Mononuclear Cells

Abstract Abstract 126 Oxygen tension regulates the function of hematopoietic stem cells (HSCs). The hypoxic microenvironment in bone marrow, known as the hypoxic niche, supports a quiescent state of HSCs and maintains long-term repopulating activity. In addition to normal HSCs, hypoxia also affects the fate of leukemic stem cells. Giuntoli et al. reported that hypoxia selects for cells with high-repopulating activity in BCR-ABL-positive leukemia cell lines that have reduced sensitivity to imatinib. The V617F-activating mutation in JAK2 plays a major role in BCR-ABL-negative myeloproliferative neoplasms (MPN). JAK2V617F activates a variety of signal transduction molecules and induces cytokine-independent growth while suppressing apoptosis in HSCs.In this study, we investigated the effects of hypoxia on JAK2V617F-positive cells. To this end, we used JAK2V617F-harboring human leukemia cell lines, HEL and SET-2. We found that culturing under hypoxic conditions (1% O2) significantly suppressed growth of these cells. Interestingly, we found that hypoxia reduced the autophosphorylation of JAK2V617F. Phosphorylation of STAT5, a major downstream target of JAK2V617F, was also highly suppressed in the hypoxic condition. Furthermore, expression of several target genes of STAT5, including cyclin D and Bcl-xL, was drastically decreased after exposure to hypoxia. For further study, after obtaining informed consent, we performed an endogenous erythroid colony (EEC) formation assay on peripheral blood mononuclear cells from JAK2V617F-positive polycythemia vera patients (n=7). We found that hypoxia suppressed EEC formation (Normoxia: 43.8±10.0; Hypoxia: 25.9±4.7, P < 0.05) and suppressed phosphorylation of JAK2 in these cells. Next, we investigated the molecular mechanisms of how hypoxia suppressed activity of JAK2V617F. To elucidate these mechanisms, we initially determined whether hypoxia induced the expression of several negative regulators for JAK2. Hypoxia did not change protein levels of SOCS1 or SOCS3, suppressors of the cytokine signaling (SOCS) family of proteins. We also confirmed that the expression of the tyrosine phosphatases PTP1B and CD45, which could dephosphorylate JAK2, were not altered in hypoxia-treated cells. In contrast, the expression of SHP-2, an SH-2 domain-containing protein tyrosine phosphatase, was drastically diminished after exposure to hypoxia in both HEL and SET2 cells. Importantly, hypoxia also suppressed the expression of SHP-2 proteins in peripheral blood mononuclear cells derived from polycythemia vera patients. To confirm that SHP-2 is required for activation of JAK2V617F, we treated HEL and SET-2 cells with an inhibitor of SHP-2. The inhibitor clearly diminished auto-phosphorylation of JAK2V617F in both cell lines. Our observations show that hypoxia suppressed activity of JAK2V617F and induced growth arrest in JAK2V617F-positive MPN cells. SHP-2 plays important roles in these processes. Interestingly, several studies have reported that SHP-2 is required for the activation of JAK2 by cytokines. In addition, recent studies have revealed that the activity of SHP-2 is increased in platelets from MPN patients. In conclusion, a hypoxic environment may modulate the fate of JAK2-positive MPN cells through suppression of SHP-2 levels and through subsequent suppression of JAK2V617F activity. Disclosures: No relevant conflicts of interest to declare.

SLIT1 Mutation in Patients with Acquired Aplastic Anemia: Its Relevance in Immune Pathophysiology

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 645-645
Author(s):  
Chizuru Saito ◽  
Kohei Hosokawa ◽  
Takamasa Katagiri ◽  
Akinori Kanai ◽  
Hirotaka Matsui ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Genetic Alterations ◽  
Receptor Expression ◽  
Healthy Individuals ◽  
Leukemia Cell Lines ◽  
Immune Mediated ◽  
Myeloid Leukemia Cell

Abstract Abstract 645 Background: Acquired aplastic anemia (AA) is a non-clonal hematopoietic disorder caused by the immune system attack on hematopoietic stem cells (HSCs) and has been thought to be rarely associated with intrinsic HSC defects. However, clonal hematopoiesis by HSCs with genetic alterations may not be uncommon in AA since clonal granulocyte populations showing the paroxysmal nocturnal hemoglobinuria (PNH) phenotype or chromosome 6p uniparental disomy (6pUPD) are detected in approximately 50% and 13% of AA patients, respectively (Katagiri T, et al, Blood, 2011). Abnormal HSCs other than those with the PIG-A mutation or 6pUPD may also contribute to hematopoiesis in AA patients due to immune escape or preferential activation of HSCs with genetic alterations in response to immune pressure in the bone marrow (BM). Objectives/methods: To identify novel genetic abnormalities in patients with AA, we carried out whole exome sequencing of peripheral blood leukocytes from 96 AA patients using a next generation sequencer. Among 40 abnormalities found in more than one patient, we selected abnormalities in genes potentially related to HSC regulation and examined the relevance of the genetic alterations to BM failure. Results: Missense mutations of SLIT1, a family of secreted glycoproteins that bind members of the Robo receptor family and regulate axon guidance, were detected in 15 of 96 patients and confirmed by Sanger sequencing in 3 patients who had been in remission for 1–3 years after immunosuppressive therapy at the time of blood sampling. Since previous studies showed that the Slit-Robo pathway negatively regulates proliferation of murine HSCs as well as a human cancer cell line in an autocrine manner (Prasad A et al., J Biol Chem; 283: 26624. 2008) and the break point of chromosome 10 in a patient with immune-mediated AA possessing t(1:10) was located within the SLIT1 gene, we studied SLIT1 and Robo receptor expression in BM mononuclear cells (BMMCs) obtained from healthy volunteers and patients with AA, and in myeloid leukemia cell lines. Although SLIT1 mRNA was scarce in un-stimulated BMMCs from healthy volunteers, SLIT1 gene expression increased 2.5–3.5 fold when incubated in conditioned medium from PHA or LPS stimulated peripheral blood mononuclear cells. The steady state expression level of SLIT1 by BMMCs from AA patients was 6 fold higher than from healthy individuals. SLIT1 mRNA was also detected in several myeloid leukemia cell lines including K562 and MV4-11, and its expression increased 2 fold by culturing the cells for 48 hours with the human stromal cell line HS-5. Examination of Robo receptor expression using antibodies specific to Robo1-4 showed that BM CD34+ cells from healthy individuals and K562 cells expressed Robo1. Recombinant Slit1 inhibited proliferation of K562 in a dose-dependent manner at concentrations of 1 and 5 mg/ml (Figure 1). Conclusions: The presence of clonal leukocytes with SLIT1 mutations in patients with immune-mediated AA suggests that HSCs with these mutations have survival or proliferation advantages over HSCs without SLIT1 mutations. Slit1 secreted from HSCs or hematopoietic progenitor cells in response to inflammatory cytokines may inhibit their own activation by binding to Robo1, but mutant Slit1 may fail to inhibit activation, leading to preferential commitment of the mutant HSCs to hematopoiesis. The Slit-Robo pathway may therefore serve as a novel therapeutic target for immune-mediated BM failure. Disclosures: No relevant conflicts of interest to declare.

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