Lifang Huang

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling

Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML chronic phase samples, about 70% of CML accelerated phase samples, and >80% CML blast crisis phase samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p < 0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p < 0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, and CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p < 0.05), implying cell-autonomous activation of Shh signaling play a predominant role in progenitor cells. Coactivation of Shh and β-catenin signaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh-neutralizing antibody or Wnt3a-neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis; however, Wnt3a peptide could salvage cell apoptosis, while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, and P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G(2)/M cell cycle arrest of progenitor cells. Our results demonstrate that autoactivated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, suggesting a novel therapeutic approach in CML.

Wnt and Notch signaling pathways selectively regulating hematopoiesis.

Hematopoietic stem and progenitor cells (HSPCs) are the source of all blood cells in the adult body. The pool of HSPCs is formed during embryogenesis process through a well-characterized succession of intra-embryonic regions and organs. The spatial and temporal restrictions in definitive hematopoietic development and the signaling molecules involved are of great interest as these may prove useful for generating and expanding these clinically important cell populations ex vivo. To elucidate the mechanism by which definitive HSPCs expand during this limited developmental time frame, we analyzed the spatial and temporal programmed gene expression patterns of Wnt and Notch signaling members during hematopoietic development. Genes related to the Wnt signaling pathway were up-regulated in E10.5 aorta-gonad-mesonephros (AGM) and E14.5 fetal liver corresponding to the inherent proliferation potential of hematopoietic progenitors, whereas genes related to the Notch signaling pathway were identified as up-regulated in E10.5 AGM, and bone marrow coincides with the maintenance of undifferentiation state of hematopoietic progenitors. Our findings suggest that Wnt and Notch signalings are integrated and are selectively regulating hematopoiesis. The spatial and temporal balance between Wnt and Notch signaling orchestrates the precise progression of hematopoietic progenitors.

Proliferation and apoptosis of bone marrow CD4+ T cells in patients with aplastic anemia and impacts of the secreted cytokines on hematopoietic stem cells from umbilical cord blood

Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from peripheral blood and bone marrow (BM) in AA patients. In the current study, BM CD4+ T cells were isolated from AA patients and healthy controls with immunomagnetic beads sorting, and proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells were compared between them. By 3H-TdR method, CD4+ T cells in AA group presented more enhanced proliferative activity. The stimulation index in control group and AA group was 1.47±0.24, and 2.51±0.34 respectively (P<0.01). After BM CD4+ T cells were induced by high concentration of CD3 monoclonal antibody for 18 h, evident apoptosis cells could be seen under the electron microscope in both control group and AA group. Flow cytometry revealed that apoptosis rates in the early and late stages of AA group were significantly higher than in control group (P<0.01). Early-stage apoptosis rate in control and AA groups was (6.85±1.48)% and (16.98±4.40)%, and late-stage apoptosis rate in control group and AA group was (2.65±1.57)% and (7.74±0.83)%, respectively (P<0.01). The CFU-GM count in AA group and control group was (74.50±9.50)/104 cells and (124.25±19.80)/104 cells respectively under an inverted microscope (P<0.01), and the expression levels of CyclinD3 mRNA and protein in cord blood CD34+ cells were both down-regulated induced by BM CD4+ T cell culture supernatant in AA patients. These results indicate that BM CD4+ T cells of AA patients are likely in an abnormally proliferative, and activated state which can correlate intimately with AA hematopoiesis damage. BM CD4+ T cells in AA patients can secret some soluble cytokines that can inhibit proliferation of hematopoietic stem cells by suppressing the expression of Cyclin D3, resulting in hematopoiesis failure.SummaryRecent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from peripheral blood and bone marrow (BM) in AA patients. In the current study, BM CD4+ T cells were isolated from AA patients and healthy controls with immunomagnetic beads sorting, and proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells were compared between them. By 3H-TdR method, CD4+ T cells in AA group presented more enhanced proliferative activity. The stimulation index in control group and AA group was 1.47±0.24, and 2.51±0.34 respectively (P<0.01). After BM CD4+ T cells were induced by high concentration of CD3 monoclonal antibody for 18 h, evident apoptosis cells could be seen under the electron microscope in both control group and AA group. Flow cytometry revealed that apoptosis rates in the early and late stages of AA group were significantly higher than in control group (P<0.01). Early-stage apoptosis rate in control and AA groups was (6.85±1.48)% and (16.98±4.40)%, and late-stage apoptosis rate in control group and AA group was (2.65±1.57)% and (7.74±0.83)%, respectively (P<0.01). The CFU-GM count in AA group and control group was (74.50±9.50)/104 cells and (124.25±19.80)/104 cells respectively under an inverted microscope (P<0.01), and the expression levels of CyclinD3 mRNA and protein in cord blood CD34+ cells were both down-regulated induced by BM CD4+ T cell culture supernatant in AA patients. These results indicate that BM CD4+ T cells of AA patients are likely in an abnormally proliferative, and activated state which can correlate intimately with AA hematopoiesis damage. BM CD4+ T cells in AA patients can secret some soluble cytokines that can inhibit proliferation of hematopoietic stem cells by suppressing the expression of Cyclin D3, resulting in hematopoiesis failure.

Fetal liver stromal cells promote hematopoietic cell expansion

Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

Tcf-1 gene silence suppresses downstream gene expression in CD4+ T cells from bone marrow of aplastic anemia patients

CD4+ T cells play a crucial role in the pathogenesis of acquired aplastic anemia (AA). Tcf-1 gene regulates T cell development and function, and it is significantly upregulated in the bone marrow CD4+ T cells from patients with acquired AA. To explore the role of Tcf-1 in the pathogenesis of AA, we knocked down Tcf-1 gene in CD4+ T cells of AA patients and studied the effects of Tcf-1 silencing on its downstream gene expression. Upon transfection of psiRNA into marrow CD4+ T cells from bone marrow of aplastic anemia patients, the expression of Tcf-1 was significantly knocked down; consequently, expressions of c-Myc and CD44 were also significantly reduced. Our results suggest that Tcf-1 may contribute to pathogenesis of AA by regulating downstream gene expression such as c-myc and CD44.

Clonal cytogenetic abnormalities are predictor in developing non-Hodgkin lymphomas?

Pathological analysis is the cornerstone for diagnosing malignant lymphoma. Status of cytogenetic abnormalities is frequently left unexamined if no evidence of malignancy is found in pathological analysis. In this study, we presented 3 cases in which clonal cytogenetic abnormalities were detected but morphological alterations of the same tissue did not support malignant non Hodgkin lymphoma at the first lymph node biopsy. Case 1 is a 55-year-old female with lymphadenopathy neoplastic process confirmed by flow cytometry and polymerase chain reaction (PCR). Chromosome analysis revealed 47,XX,t(3;22)(q27;q11),+del(9)(p12)[16]/46,XX[4]. The pathological analysis of subsequent lymph node biopsy indicated diffuse large B-cell lymphoma (DLBCL). Case 2, a 74-year-old female, for whom the pathological analysis, molecular studies and flow cytometric analysis of the first lymph node biopsy found no evidence of clonal cell. Cytogenetic analysis demonstrated a terminal deletion of chromosome 7 and 1, and the patient received a second lymph node biopsy and splenectomy. A pathological diagnosis of splenic marginal zone lymphoma (SMZL) was made. In Case 3 who was a 66-year-old female with right cervical and axillary lymph node enlargement. Cytogenetic analysis showed clonal karyotypic abnormalities: 48,XX, t(14;18)(q32;q21) [13]/46, XY [7]. The diagnosis of follicular lymphoma was rendered by the second biopsy of axillary lymph node according to the analysis of morphology and immunohistochemistry. We propose that clonal cytogenetic abnormalities may be a high potential risk for developing non-Hodgkin lymphomas. Follow-up and rebiopsy must be performed in patients who are cytogenetically abnormal but morphologically benign.

Cytogenetics with flow cytometry in lymph node/extranodal tissue biopsies is sensitive to assist the early diagnosis of suspected lymphomas

Few studies have examined the value of cytogenetic studies with flow cytometry (FC) in lymph node/extranodal tissue biopsies with suspected lymphoma. To evaluate this, G-banded karyotyping and/or fluorescence in situ hybridization (FISH) with FC immunophenotyping were performed on 185 lymph node or extranodal tissue biopsy specimens with suspected lymphoma. Complete cytogenetic analysis of lymph node/extranodal tissue was successful in 174 cases (94.1%) and 57.5% demonstrated chromosomal abnormalities. In 116 malignant lymphoma cases, 83.8% showed abnormalities. In 74 B cell lymphomas (B-NHL), abnormalities were more frequent in lymph node/extranodal tissues than in bone marrow by conventional cytogenetics (CC, 97.2 vs 26.1%), FISH (70.6 vs 17.6%), and FC (98.6 vs 28.4%). Three B-NHL diagnoses were confirmed by re-biopsy of lymph nodes due to the presence of abnormalities in the first biopsy, but no evidence of malignancy in pathological, FC, or IgH/TCR gene rearrangement analyses. In 29 T cell lymphomas (T-NHL), abnormalities were more frequent in lymph nodes than in bone marrow by CC (67.9 vs 21.4%) and FC (75.9 vs 27.6%) analyses. As expected, in 13 Hodgkin lymphoma cases, abnormalities were more frequent in lymph nodes than bone marrow by CC (41.7 vs 16.7%) and FC (30.8 vs 7.7%) analyses. In 56 reactive lymphoid hyperplasias (RLH), 7.1% had conventional clonal cytogenetic abnormalities. Two of these patients died of disease progression and two had their pathological diagnosis revised after the second review. These findings indicate that cytogenetic analysis combined with FC in lymph node/extranodal tissue biopsies can provide critical information in the auxiliary diagnosis of lymphoma.

In vitro effects of imatinib on CD34 + cells of patients with chronic myeloid leukemia in the megakaryocytic crisis phase

Imatinib is a tailored drug for the treatment of chronic myeloid leukemia (CML), and has substantial activity and a favorable safety profile when used as a single agent in patients with CML in myeloid blast crisis. The megakaryocytic blast crisis in CML occurs rarely and carries a poor prognosis. The aim of the present study was to investigate the effects of imatinib on cluster of differentiation (CD)34+ cells from patients with CML in the megakaryocytic crisis phase. Bone marrow mononuclear cells (BMNCs) were isolated from patients with CML in the megakaryocytic crisis phase. CD34+ cells were selected from BMNCs by positive immunomagnetic column separation. Imatinib significantly induced G1 arrest, reduced the phosphorylation of cyclin-dependent kinase 1 and retinoblastoma proteins and inhibited the proliferation of CD34+ cells from patients with CML in the megakaryocytic crisis phase. Annexin V/propidium iodide and caspase-3 activity showed that imatinib induced apoptosis. Western blot analysis and protein tyrosine kinase activity assays showed that imatinib inhibited BCR-ABL protein tyrosine kinase activity. The in vitro data thus markedly indicate a potential clinical application of imatinib for patients with CML in the megakaryocytic crisis phase.