Role of hERG1 K+ Channels as a Positive Regulator In SDF-1alpha-Mediated Proliferation of Leukemia Cells and Leukemia Stem/Progenitor Cells

Abstract Abstract 4786 Background: Mounting evidence that leukemia stem cells (LSCs) occupy and receive important signals from specialized areas (“niches”) that alter the stromal microenvironment and disrupt normal hematopoiesis. The innovative therapeutic strategies focus on targeting of microenvironmental interactions in leukemia. Therefore, it is important to fully elaborate the mechanisms of microenvironment- mediated leukemogenesis. Stromal-cell derived factor-1alpha (SDF-1à) is the main cytokine produced by bone marrow stromal cells. The SDF-1à/CXCR4 axis specifically mediates homing and migration of leukemic blasts. While our previous work has shown that SDF-1à significantly increases hERG1 K+ tail current and a specific hERG1 K+ channels inhibitor significantly blocks SDF-1à- induced migration of leukemic cells. In fact, recent studies suggested that the human ether à-go-go-related gene (HERG) K+ channels are constitutively expressed in AML stem/progenitor cells, and regulate cell proliferation as well as clinical prognosis. Here we investigate the hypothesis that a new leukemic blast–stromal interaction is mediate by hERG1 K+ channels and SDF-1à. Methods: Proliferation assay, apoptosis and cell cycle analysis were used to analyze effects of E-4031(a specific hERG1 K+ channels inhibitor) in the presence of SDF-1à on leukemia cell lines HL-60. RT–PCR and western blot analysis were used to determine changes in herg1 expression and Wnt/β-catenin signaling pathway in response to SDF-1à in the presence and absence of E-4031. Primary leukemias obtained from the bone marrow of de novo AML patients (n=6) at diagnosis. Mononuclear cells were isolated from the samples using Ficoll-Paque density gradient separation, and cultured with SDF-1à in the presence and absence of E-4031. AML colony-forming cell (CFC) assays and flow cytometry were performed to assess the effects of E-4031 in the presence of SDF-1à on LSCs. Results: SDF-1a enhanced cell proliferation in a dose-dependent manner. The maximal increase by 1.6 times was obtained for 100ng/ml. While this effect was impaired by E-4031, which significantly impaired cell proliferation induced by SDF-1a with a concentration of 100ng/mL by (40.3±8.4)%. In addition, E-4031 inhibited SDF-1a-stimulated leukemic cell proliferation by inducing G0/G1 arrest. Cell apoptosis analysis revealed that either E-4031 or SDF-1a has direct effect on HL-60 cell apoptosis. Unexpected, there was no significant synergistic effect upon apoptosis. After exposures to 100ng/ml SDF-1à, hERG1 mRNA and protein levels increased significantly, by approximately 1.5-fold above control levels. Moreover, SDF-1a increased the expression of Wnt/β-catenin target genes, including β-catenin, cyclin-D1, and c-myc. Interestingly, this manner was abolished by E-4031. The presence of progenitor cells was evaluated by plating suspension cells cultured with SDF-1a in CFC assays. E-4031 decreased numbers of CFC in suspension to 77.3%. Upon expansion with SDF-1a, E-4031 resulted in a significant reduction in the number of progenitors to 31.8%. The effects on LSCs were determined on phenotypically described stem cells from AML. Treatment with 1μ M E-4031 for 48 hours inhibited the proliferation of LCSs compared with untreated controls, a mean viability of 11.8% for CD34+CD38- and 10.4% for CD34+CD38+. In contrast, a significant decrease in the viability of stem cells after E-4031 in the present of SDF-1a treatment, with only 9.6% for CD34+CD38- and 9.5% for CD34+CD38+. Conclusions: Initial studies provided evidence that the hERG1 K+ channels and SDF-1 emerged as mediators of stromal/leukemic cell interactions, which largely contribute to the proliferation mediated by the microenvironment. Likewise, other components of bone marrow microenvironment, such as Wnt/β-catenin signaling pathway, may modulate hERG1 K+ channels in leukemic cells. Taken together, these results provided rationale for studies of new molecular events involved in bone marrow microenvironment and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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The Chemokine Receptor CXCR-4 Is Expressed on CD34+Hematopoietic Progenitors and Leukemic Cells and Mediates Transendothelial Migration Induced by Stromal Cell-Derived Factor-1

Blood ◽

10.1182/blood.v91.12.4523 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4523-4530 ◽

Cited By ~ 459

Author(s):

Robert Möhle ◽

Frank Bautz ◽

Shahin Rafii ◽

Malcolm A.S. Moore ◽

Wolfram Brugger ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cell ◽

Leukemic Cell ◽

Transendothelial Migration ◽

Leukemic Cells ◽

Hematopoietic Progenitor ◽

Leukemic Cell Lines ◽

Stromal Cell Derived Factor

Abstract The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1–deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1–induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38− and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1–induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1β induced migration of CXCR-4–positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.

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The Chemokine Receptor CXCR-4 Is Expressed on CD34+Hematopoietic Progenitors and Leukemic Cells and Mediates Transendothelial Migration Induced by Stromal Cell-Derived Factor-1

Blood ◽

10.1182/blood.v91.12.4523.412k04_4523_4530 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4523-4530 ◽

Cited By ~ 7

Author(s):

Robert Möhle ◽

Frank Bautz ◽

Shahin Rafii ◽

Malcolm A.S. Moore ◽

Wolfram Brugger ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cell ◽

Leukemic Cell ◽

Transendothelial Migration ◽

Leukemic Cells ◽

Hematopoietic Progenitor ◽

Leukemic Cell Lines ◽

Stromal Cell Derived Factor

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1–deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1–induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38− and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1–induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1β induced migration of CXCR-4–positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.

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Survivin Is Highly Expressed in AML Stem Cells and Predicts Poor Clinical Outcome

Blood ◽

10.1182/blood.v118.21.238.238 ◽

2011 ◽

Vol 118 (21) ◽

pp. 238-238

Author(s):

Bing Z Carter ◽

Yihua Qiu ◽

Xielin Huang ◽

Lixia Diao ◽

Nianxiang Zhang ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Cox Model ◽

Survivin Expression ◽

Leukemic Cells ◽

White Blood Count ◽

Expression Levels ◽

Mutational Status

Abstract Abstract 238 Survivin, a member of the inhibitor of apoptosis (IAP) protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignant cells, including leukemic cells. We showed previously that targeting survivin expression in AML cells induces a cell proliferation defect and subsequent cell death involving the mitochondrial pathway. To assess its usefulness as a prognostic marker in AML, we profiled survivin expression in samples from 511 newly diagnosed AML patients using a validated reverse-phase protein array and correlated its protein expression levels with clinical outcomes. We found that survivin levels were higher in the bone marrow (BM) than in peripheral blood (PB) leukemic cells in 140-paired samples (p = 0.0001) consistent with its function in cell proliferation. Survivin levels did not correlate with cytogenetic groups, the mutational status of nucleophosmin or FLT-3, BM or PB blast counts, white blood count, or patient performance status. Interestingly, survivin levels were found to significantly predict shorter overall (P = 0.016) and event-free (P = 0.023) survival in multivariate Cox model analysis. Age, gender, white blast count, BM blasts, cytogenetics, and survivin levels were included in the final model. The groups expressing low and high survivin were defined by the median value. This was also true when data sets from BM and PB were analyzed separately. Further, we found that survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk AML blasts and total CD34+ cells (2.06 and 1.91 fold, respectively, P < 0.001) when we analyzed survivin expression in 37 samples of isolated CD34+38− AML cells (see Figure). These results suggest that survivin expression levels are prognostic in AML and that survivin is overexpressed in AML stem/progenitor cells, may play an important role in AML stem cells, and may thus be an important target in AML therapy. Disclosures: No relevant conflicts of interest to declare.

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Exosomes Derived from AML/MDS Cells Is Involved in Stromal Dysfunction and Bone Marrow Failure

Blood ◽

10.1182/blood.v124.21.4627.4627 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4627-4627

Author(s):

Hiroto Horiguchi ◽

Masayoshi Kobune ◽

Shohei Kikuchi ◽

Wataru Jomen ◽

Kazuyuki Murase ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mrna Expression ◽

Progenitor Cells ◽

Stromal Cells ◽

Leukemic Cells ◽

Boyden Chamber ◽

Hematopoietic Stem ◽

Coculture System ◽

Qpcr Array

Abstract The failure of normal hematopoiesis in myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) could be induced by a variety of mechanism such as the alteration of property of hematopoietic stem cells and stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells especially regarding involvement of mesenchymal stromal cells (MSCs). To gain insight into the mechanism of stromal dysfunction, comparative analyses of transcriptomes were conducted between normal and MDS/AML-derived MSCs. Further, we attempted to identify certain effectors originated from MDS/AML cells could alter the function of bone marrow (BM) MSCs. The MSCs derived from healthy volunteer (HV)-derived (normal) and MDS/AML-derived stromal cells were established and analyzed mRNA expression by quantitive PCR (qPCR) array. Additionally, the supporting activity of MSCs for BM CD34+ progenitor/stem cells was examined using serum free coculture system. The interaction between MDS/AML cells and MSCs were evaluated by using Boyden Chamber and the changes of mRNA expression were analyzed. The results of qPCR array revealed that the expression of hematopoietic factors was drastically altered in MDS/AML-derived MSCs as compared with normal MSCs. Among these factors, the expression of SCF and JAG1 mRNA were significantly and consistently reduced in all MDS/AML patients examined. Functional assay of these MSCs demonstrated that the number of colony-forming units (CFU) mixed cells (MIXs) and cobblestone area-forming cells (CAFCs) derived from CD34+ cells was significantly reduced after coculture with MDS/AML-derived MSCs as compared with normal MSCs. Even non-contact culture using Boyden Chamber between leukemic cells and MSCs induced the reduction of SCF and JAG1 mRNA, indicating that certain inducers could be soluble factors. Interestingly, this effect of transcriptomes alteration was negated by nSMase2 inhibitor (GW4869). Exosome transfer assay using Boyden Chamber revealed that GFP and PKH26 in leukemic cells transmit onto MSCs in non-contact coculture system and this transfer of exosome was significantly inhibited by GW4869 or nSMase siRNA. The multiple type of microRNA in exosome derived from MDS/AML cells was transferred into MSCs, suggesting that exosome could contribute to the alteration of mRNA expression in stromal cells. Collectively, these results indicated that exosome derived from MDS/AML cells could be involved in the reduction of SCF/JAG mRNA and the stromal supporting activity of normal hematopoietic stem/progenitor cells. Disclosures No relevant conflicts of interest to declare.

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Oncogene Cooperativity Analysis Reveals a Novel Set of Genes That Regulate the In Vivo Growth and Survival of Leukemia Stem Cells

Blood ◽

10.1182/blood.v118.21.553.553 ◽

2011 ◽

Vol 118 (21) ◽

pp. 553-553

Author(s):

John M Ashton ◽

Marlene Balys ◽

Sarah Neering ◽

Glenn Cowley ◽

David E. Root ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Leukemic Cell ◽

Gene Signature ◽

Leukemic Cells ◽

Leukemia Stem Cells ◽

Human Leukemia ◽

Normal Cells ◽

In Vivo Growth

Abstract Abstract 553 In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia. Disclosures: No relevant conflicts of interest to declare.

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Application of hyperthermia to the treatment of human acute leukemia: purging human leukemic progenitor cells by heat

Blood ◽

10.1182/blood.v67.3.802.802 ◽

1986 ◽

Vol 67 (3) ◽

pp. 802-804 ◽

Cited By ~ 39

Author(s):

Y Moriyama ◽

M Narita ◽

K Sato ◽

M Urushiyama ◽

S Koyama ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Progenitor Cells ◽

Autologous Bone Marrow Transplantation ◽

Leukemic Cell ◽

Autologous Bone Marrow ◽

Leukemic Cells ◽

Neoplastic Disease ◽

Myeloid Progenitor Cells

Abstract The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.

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Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

Blood ◽

10.1182/blood.v116.21.4836.4836 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4836-4836

Author(s):

Gyeongsin Park ◽

Michael Heuser ◽

Tobias Berg ◽

R. Keith Humphries

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Fixed Number ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

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Distinct Effects of SCF and Hes1 On Normal Hematopoietic Stem and Progenitor Cells During Leukemic Cell Expansion in a T-ALL Mouse Model

Blood ◽

10.1182/blood.v120.21.1216.1216 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1216-1216

Author(s):

Chen Tian ◽

Zhipan Cao ◽

Qiao Li ◽

Jinhong Wang ◽

Zhenyu Ju ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Real Time ◽

Hematopoietic Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Rt Pcr ◽

Hematopoietic Stem

Abstract Abstract 1216 During leukemia development, emerging leukemic cells out-compete normal hematopoietic cells and become predominant in the body. How hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) respond to the growth of leukemic cell population is an important, yet less investigated area. Our previous study demonstrated differential effects of a leukemic environment on normal HSCs and HPCs in the Notch1-induced T-ALL mouse model (Hu X, et al. Blood 2009). We found that normal HSCs were better preserved in the leukemic bone marrow in part due to increased quiescence of the HSCs and in contrast, HPCs were exhausted during the expansion of leukemic cells. Our current work is aimed to further explore the molecular mechanisms concerning the distinct impacts of leukemic environment on normal HSCs and HPCs in the T-ALL mouse model. Given the previous report by others showing that increased secretion of stem cell factor (SCF) by myeloid leukemia cells played an important role in inducing normal HSCs/HPCs out of their niche and thus allowing leukemic cells to occupy the niche in the human-NOD/SCID xeno-graft model (Sipkins DA et al, Science 2008), we first examined the expression of SCF by ELISA, Western blot and real-time RT PCR in both normal hematopoietic and leukemic cell fractions in the Notch1-induced T-ALL mouse model as previously reported. We found that while expression of SCF in peripheral blood (PB) or bone marrow (BM) was increased in the leukemic mice, both mRNA and protein levels of SCF in normal hematopoietic cells were higher than that in leukemic cells, thereby suggesting that elevated SCF might be mainly secreted by non-leukemic cells in the leukemic hosts of our model. Further assessments on the role of SCF in leukemogenesis with the mice specifically deficient in SCF in different niche cell types are currently under investigation in our laboratory. In order to define potential mediators in HSCs in response to leukemic cell growth, a microarray study on normal HSCs isolated from T-ALL leukemic mice and the control mice was conducted. Gene expression profiling showed significantly differed expression of 169 genes (127 up and 42 down). Especially, real-time RT PCR confirmed an increase of Hes1, p21, Fbxw11, IL-18R1 and Itgb3, and a decrease of CXCR4 and Mmp2. Interestingly, the expression of Hes1 and its target gene, p21 were elevated in normal HSCs but not in HPCs, letting us to hypothesize that Hes1 might be in part mediate the different responses of HSCs and HPCs to the T-ALL leukemic environment. To test this hypothesis, we ectopically expressed Hes1 in normal hematopoietic cells and then examined their functions under the leukemic condition. BM cells from B6.SJL mice were transduced with either MSCV-Hes1-IRES-GFP or control MSCV-GFP vector. After transduction, Hes1-GFP+or control-GFP+cells were co-transplanted with the Notch1-induced T-ALL cells into lethally irradiated C57BL/6J recipients. The engrafted cells from the leukemic BM were analyzed and Hes1-GFP+or control-GFP+cells were sorted for functional assessments. Interestingly, although over-expression of Hes1 inhibited the growth of colony forming cell (CFC) in vitro, it could potentiate the long-term repopulating cells by maintaining more cells in the quiescent (G0) state in vivo. Taken together, our current study supports a role of Hes1 in mediating the distinct responses of normal HSCs and HPCs to the T-ALL leukemic environment. Disclosures: No relevant conflicts of interest to declare.

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Effect of 25-Hydroxycholesterol on the Differentiation of Bone Marrow Mesenchymal Stem Cells Through Wnt5/TGF-β Signaling Pathway

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2172 ◽

2019 ◽

Vol 9 (11) ◽

pp. 1583-1588

Author(s):

Shaoting Li ◽

Jinhe Zhou ◽

Zhiqing Ye ◽

Shenglin Wu

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Signaling Pathway ◽

Caspase 3 ◽

Control Group ◽

Nodule Formation ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) can be multi-directionally differentiated and are widely used in tissue engineering. 25-hydroxycholesterol (25-HC) can induce osteogenesis and is involved in osteogenic formation. However, the role of 25-hydroxycholesterol in BMSCs is unclear. Rat BMSCs were isolated and divided into control group and 25-HC treatment (2 and 4 μM) group. Cell proliferation was detected by MTT assay. Caspase-3 and ALP activity was analyzed. Real time PCR was done to analyze Runx2, OPN, FABP4 and PPARγ2 expression. Red staining detects the calcified nodule formation. Wnt5 level was detected by western blot and TGF-β secretion was analyzed by ELISA. 25-HC treatment significantly inhibited cell proliferation, increased Caspase 3 activity, decreased ALP activity and the expression of Runx2 and OPN, increased expression of FABP4 and PPARγ2, decreased formation of calcified nodules, secretion of TGF-β and reduced expression of Wnt5 compared to control group (P < 0.05), and the above changes were significant with the increase of the concentration of 25-HC (P < 0.05). 25-hydroxycholesterol regulates the proliferation and apoptosis of BMSCs by regulating Wnt5/TGF-β signaling pathway, inhibiting the differentiation of BMSCs into osteogenic direction and promoting its adipogenic differentiation.

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Increased Expression of HERG1 K+ Channels Contribute to MDS Progression and Display Correlation with Prognosis Stratification

Blood ◽

10.1182/blood.v126.23.5193.5193 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5193-5193

Author(s):

Li Lu ◽

Wen Du ◽

Wei Liu ◽

Dongmei Guo ◽

Shiang Huang ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Cell Proliferation ◽

Therapeutic Target ◽

Mononuclear Cells ◽

Refractory Anemia ◽

Potential Therapeutic Target ◽

K Channels

Abstract Background: Myelodysplastic syndromes (MDS) are defined as a heterogeneous group of clonal hematopoietic stem cell (HSC) malignant disorders which are characterized by bone marrow failure and dysplasia of blood cells. As continually terminated in AML, MDS are treated as the "pre-leukemia" condition. HERG K+ channels, being three subtypes: HERG1, HERG.2, HERG3, are expressed transiently at early stages of cells such as progenitor and stem cells, disappearing at later stages of cells like mature cells. Our previous work and others demonstrated that HERG1, as an oncoprotein, was over expressed in AML cells and played crucial roles in SDF-1induced leukemia cell migration. The expression and functional role of HERG1 K+ channels in MDS development is not reported. We investigated the HERG1 K+ channels expression and explored the functional link between HERG1 K+ channels and MDS progression Methods: The expression of HERG1 K+ channels in untreated MDS, AML patients and normal control was detected by flow cytometry. The roles of HERG1K+ channels in regulation of SKM-1 cell proliferation, apoptosis and cell cycle by CCK-8 assay and flow cytometry, respectively. Results: We observed that the expression of HERG1 K+ channels on bone marrow (BM) mononuclear cells (MNCs) in MDS patients was significantly higher than that in the controls (42 ± 7.62% vs 19.8 ± 2.79%, p < 0.01) , but was lower than that in AML (42 ± 7.62% vs 52.18 ± 9.72%, p < 0.01). MDS subtypes mainly contained refractory cytopenia unilineage dysplasia (RCUD), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blast I (RAEB-I), refractory anemia with excess blasts II (RAEB-II) and MDS-unclassified (MDS-U). We next analyzed percentage of HERG1 K+ channels in MDS subtypes and found that level of HERG1 K+ channels on the MNCs in each subtypes of MDS was significant higher than that in the control group (Control: 19.8 ± 2.79%, MDS-RCUD: 30.91 ± 1.48%, MDS-RCMD: 39.06 ± 2.47%, MDS-RAEB-I: 44.76 ± 5.54%, MDS-RAEB-II: 49.69 ± 3.28%, p < 0.01), suggesting that HERG1 K+ channels expression might be positively associated with malignancy degree of MDS. HSCs played important roles in the pathophysiology of MDS. Our results also revealed that with the increase of malignancy degree, the percentage of HERG1K+ channels on CD34+CD38- derived cells from MDS subtypes tended to elevate in corresponding MDS subtypes (MDS-RCUD: 61.21 ± 9.46%, MDS-RCMD: 65.66 ± 4.57%, MDS-RAEB-I: 72.35 ± 9.38%, MDS-RAEB-II: 75.71 ± 4.24%, p < 0.05), which supported the notion in other way that MDS are HSC malignant disorders. The over-expression of HERG1 K+ channels on CD34+CD38- cells in MDS patients might be correlated with the oncogenesis of MDS. In addition, the prognosis stratification of MDS patients was performed according to International Prognostic Scoring System (IPSS) scores and the untreated MDS cohort was categorized as four risk groups: Low-R, Int-1-R, Int-2-R and High-R. Our results showed that there was a positively correlation between HERG1 level and IPSS scores of patients (Low-R: 31.93 ± 3.47%, Int-1-R: 39.95 ± 5.76%, Int-2-R: 45.94 ± 6.34%, High-R: 49.05 ± 3.04%, p < 0.01). This showed that expression level of HERG1 K+ channels was helpful for predicting the prognosis of de novo MDS. Furthermore, we analyzed HERG1 K+ channel role on MDS cell proliferation and apoptosis. Incubation with 0, 10, 20 uM E-4031 ( HERG K+ channels inhibitor) with SKM-1 cells (MDS cell line) for 48 h, the results showed that blockage of HERG1 decreased the proliferation of SKM-1 cells but had rarely effects on cell apoptosis and cell cycle distribution. In consistent with other studies, HERG1 K+ channels had already been shown to be necessary for growth of cancer cells through specific activities independent of cell cycle such as interacting with TNFR1 protein which could activate NF-κB to facilitate cell proliferation and favoring transduction of growth signals by MAP kinase/c-fos pathway. These findings showed that physiological activity of HERG1 K+ channels was crucial for MDS cell proliferation and HERG1 K+ channels may be a potential therapeutic target for MDS. Conclusion: Briefly, our study firstly showed that HERG1 K+ channels were aberrantly over-expressed on MDS stem cells, mononuclear cells, and positively associated with malignancy degree of MDS. HERG1 K+ channels functionally contribute to MDS progression and may be a potential therapeutic target for MDS. Disclosures No relevant conflicts of interest to declare.

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