AMD3100 and G-CSF disrupt the cross-talk between leukemia cells and the endosteal niche and enhance their sensitivity to chemotherapeutic drugs in biomimetic polystyrene scaffolds.

BACKGROUND: The multidrug resistance of leukemia cells is closely related to the microenvironment. The present leukemia microenvironment models focus on two-dimensional co-culture system in vitro which does not mimic the in vivo cell growth, while the 3D polystyrene (PS) scaffolds have the advantage. Stromal cell derived factor-1 may be involved in the shielding of endosteal niche from leukemia cells by binding to its receptor CXCR4, but the relationship between SDF-1/CXCR4 axis and leukemia cells is unclear. DESIGN AND METHODS: The experiments were built on the 3D PS scaffolds coated with osteoblasts. Stromal cells and MV4-11 cells were plated on the scaffolds. Then G-CSF, AMD3100 and cytarabine were added. Adhesive rate, SDF-1 level, migration state, apoptosis rate, and cell cycle of leukemia cells were observed after incubation at 24h and 48h. RESULTS: G-CSF decreased the level of SDF-1 and inhibited the expression of CXCR4 and promoted stationary phase leukemia cells to enter the mitotic phase and enhanced the killing effect of chemotherapeutic drugs. AMD3100 disrupted the interaction between tumors and matrix, mobilized the leukemia cells to keep away from the protective microenvironment and strengthened the cytotoxic effect of Ara-C. The combination of G-CSF and AMD3100 had stronger effects on killing the leukemia cells induced by Ara-C. CONCLUSION: It demonstrates that AMD3100 and G-CSF may inhibit adhesion and migration abilities of leukemia cells with the bone marrow niche. Both of them inhibit the role of SDF-1/CXCR4 directly or indirectly. Thus inhibiting SDF-1/CXCR4 axis may be helpful to the treatment of refractory AML.

High-dose methotrexate in the mobilization of hematopoietic stem cells for patients with non-Hodgkin's lymphoma: a twelve-year study in a single center.

BACKGROUND: High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) is a promising approach for non-Hodgkin's lymphoma (NHL). Higher cell doses have been associated with a faster blood count recovery and a reduction in transfusion requirements, infection rates, and hospitalization times. Mobilization failure constitutes one of the main reasons for avoiding auto-HSCT. The role of high-dose methotrexate (MTX) as mobilization regimen is still unclear. STUDY DESIGN AND METHODS: The effect of high-dose MTX as a mobilization regimen for 67 adult patients with NHL who received auto-HSCT was studied between January 2001 and October 2012. The stem cells were mobilized using combination chemotherapy including MTX plus granulocyte-colony-stimulating factor (G-CSF) in 33 patients (Group A), and the stem cells of the other 34 patients were mobilized using the same combination chemotherapy plus G-CSF without MTX (Group B). RESULTS: All of the patients were successfully mobilized in Group A; however, two patients failed in Group B. The median numbers of CD34+ cells collected were 14.36 × 10(6) and 5.3 × 10(6) cells/kg for Groups A and B, respectively (p < 0.05). All of the patients experienced a stable neutrophil and platelet (PLT) engraftment. The times to white blood cell engraftment were 8.0 days in Group A and 11.0 days in Group B, and the times to PLT engraftment were 12.0 days in Group A and 13.0 days in Group B (p < 0.05 for both variables). CONCLUSION: High-dose MTX is a powerful regimen component for stem cell mobilization in adult patients with NHL.

[Clinical Efficiency of Rituximab Combined with Autologous Hematopoietic Blood Stem Cell Transplantation for Treatment of CD20+ B non.Hodgkin Lymphoma].

AbstractObjective: To investigate the effeciency of autologous hematopoietic stem cell transplantation (auto.HSCT) combined with rituximab(R) to treat CD20+ B non.Hodgkin lymphoma(B.NHL). METHODS: From January 2005 to December 2013, 83 patients with refractory/recurrent CD20+ B.NHL who were treated with auto.HSCT in our department were enrolled. The patients were divided into 2 groups: 57 patients in Rituximab group, and 26 patients in control group(without Rituximab). All the patients received chemotherapy and auto.HSCT. For the patients in treatment group, Tituximab was used before transplantation of the stem cells, and for some patients Rituximab was used after transplantation. For the patients in control group, the induction, enhancement and transplantation were the same as those in treatment group. The clinical efficiency of the patients in treatment group according to the time and frequency of R was analyzed in subgroups and compared with the control group. The deadline of follow.up was April 30 2014. RESULTS: All the patient achieved complete response. The median follow.up time was 39 months. Both the two groups collected peripheral blood stem cells successfully, and had no difference in hematopoietic reconstitution time. Three patients in treatment group and six patients in control group relapsed and the three year overall survival and EFS in treatment group was significantly higher than that in control group, that is（93.0% vs 73.1%, P=0.037） and （89.5% vs 65.4%, P=0.034）, respectively. Subgroup analysis showed that: compared with the treatment group in which using R in the whole courses(before and after transplantation, and collection of stem cells) was superior to the control group in both OS and EFS, with the OS 97% vs 87.5% (P>0.05) and EFS 97% vs 76.2% (P=0.05) respectively. While stratified by the different courses of rituximab, the OS was 88.9% (1-2 courses, 9 cases), 93.1% (3-4 courses, 29 cases), 94.7%(more than 5 courses,19 cases), and EFS was 77.8%, 89.7% and 94.7%, respectively. CONCLUSION: For the patients with refractory/recurrent CD20+ B.NHL, the combination of R and inducing chemotheraphy, purify in body before transplantation, as well as continue with R after auto.HSCT could obviously improve the OS and EFS of patients. For the patients who with R before and after transplantation, their EFS is better than the patients with R before transplantation only.

Cost and outcome in stem cell collections in HLA-haplo identical/mismatched related transplantation with combined granulocyte-colony stimulating factor-mobilized blood and bone marrow for patients with hematologic malignancies.

Unmanipulated HLA-haplo identical/mismatched related transplantation with combined granulocyte-colony stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) and granulocyte-colony stimulating factor-mobilized bone marrow (G-BM) has been developed as an alternative transplantation strategy for patients without an HLA-matched related or unrelated donor. In this transplantation setting, the cost and outcome of stem cell collections have not been defined completely. The aim of this study was to compare the cost and outcome of stem cell collection in HLA-haplo identical/mismatched related transplantation with combined G-PBSCs and G-BM to the HLA-identical/matched transplantation with G-PBSCs alone for patients with hematologic malignancies. Hundred and fifty-two healthy donors received twice-daily granulocyte-colony stimulating factor (G-CSF) subcutaneously for 5 days. The PBSCs were collected on day 4 and 5 of G-CSF treatment for HLA-identical/matched transplantation from unrelated/related donors. The PBSC collections and BM harvests was performed on day 4 and 5 of G-CSF treatment for HLA-haplo identical/mismatched related transplantation from related donors, respectively. There was no difference in the major characteristics between groups. More stem cells were harvested in HLA-haplo identical/mismatched related donors than that of HLA-identical/matched donors and a lower cost was seen in the former. The HLA-haplo identical/mismatched related transplantation with combined G-PBSCs and G-BM was a feasible approach with high cell harvest and low cost of stem cell collection for patients with hematologic malignancies.

Role of antithymocyte globulin and granulocyte-colony stimulating factor-mobilized bone marrow in allogeneic transplantation for patients with hematologic malignancies.

The main obstacle for allogeneic transplantation is delayed hematologic reconstitution and serious graft-versus-host disease (GVHD). The results of 128 patients with hematologic malignancies undergoing HLA-identical (n=52) or HLA-haploidentical/mismatched (n=76) hematopoietic stem cell transplantation (HSCT) performed during the same time period were compared. Patients with HLA-identical HSCT received unmanipulated granulocyte-colony stimulating factor-mobilized peripheral blood stem cells (G-PBSCs). Forty-six patients with HLA-haploidentical related HSCT received antithymocyte globulin (ATG) in conditioning regimens followed by the transplantation of the combination of unmanipulated G-PBSCs and granulocyte-colony stimulating factor-mobilized bone marrow (G-BM) and 30 patients with HLA-mismatched unrelated HSCT received ATG in conditioning regimens followed by the transplantation of unmanipulated G-PBSCs. All patients got successful hematopoietic engraftment. The cumulative incidences of grades I to II acute GVHD (aGVHD) on day 100 in the identical, haploidentical related and mismatched unrelated cohorts were 21.2%, 43.5%, and 53.3%, respectively. The cumulative incidences of chronic GVHD (cGVHD) in the identical, mismatched unrelated, and haploidentical related cohorts were 34.6%, 33.3%, and 10.9%, respectively. The 2-year relapse and treatment-related mortality (TRM) rates were 19.2%, 23.9%, 23.3%, and 9.6%, 8.7%, 10% for patients who underwent identical, HLA-haploidentical related, and mismatched unrelated transplantation, respectively. The 2-year probabilities of leukemia-free survival and overall survival were 72.2%, 70.6%, 68.1%, and 76.5%, 77.8%, 70.0% after identical, haploidentical related and mismatched unrelated transplantations, respectively. Multivariate analyses showed that only advanced disease stage and a diagnosis of disease had increased risk of relapse, treatment failure, and overall mortality. In conclusion, it is a feasible approach with acceptable outcomes for patients undergoing HLA-haploidentical related HSCT by the combination of G-PBSCs and G-BM with conditioning regimens including ATG.

[Exploratory study of novel leukemia bone marrow stromal cell adhesion mediated drug resistance model].

OBJECTIVE: To construct cell adhesion mediated drug resistance (CAM-DR) model based on Acute lymphocyte leukemia bone marrow stromal cells(BMSC) for further studying drug resistance of leukemia. METHODS: Firstly, we adhesively cultured Jurkat cell strain of human leukaemia lymphocyte with the matrix cell radiated by (60)Co to construct the model of CAM-DR, then evaluated the model in morph and construction by scanning electron microscope. The IC50 of DNR on Jurkat cell was examen by MTT and the concentration and distribution of DNR in the cell was detected by flow cytometry. RESULTS: When the Jurkat cells were cultured with BMSC for 24 h, we found that Jurkat cells had adhesioned the bone marrow stromal cell layer by parapodium and some of them had nichd the mesh consisted by the confluence of BMSC. At the point of 48 h, some Jurkat cells had migrated to underlayer of BMSC, and Jurkat cells were nichd the mesh of BMSC like nidi. The accumulation and distribution of DNR in the Jurkat cells were not affected in the model, but the reaction of Jurkat cells to DNR were significantly inhibited. CONCLUSION: The model of CAM-DR based on Acute lymphocyte leukemia bone marrow stromal cells was successfully built.

Targeting of the leukemia microenvironment by c(RGDfV) overcomes the resistance to chemotherapy in acute myeloid leukemia in biomimetic polystyrene scaffolds.

The bone marrow microenvironment provides a relative sanctuary from cytotoxic drugs for leukemia cells. The present niche models concentrate on a two-dimensional (2D) co-culture system in vitro, which does not imitate the in vivo environment, while the 3D scaffolds are more reflective of this. Osteopontin (Opn) secreted by bone marrow osteoblasts, may participate in protecting leukemia cells from apoptosis by binding to its receptor αvß3, which can be expressed on the surface of the leukemia MV4-11 cell line. However, the association between the Opn/αvß3 axis and leukemia cells is unknown. In the present study, experiments were conducted on 3D polystyrene scaffolds coated with osteoblasts and leukemia cells. The cells were exposed to cyclo(Arg-Gly-Asp-d-Phe-Val) [c(RGDfV)] (35 nmol/ml), which blocks αvß3, for a period of 24 h. Cytarabine was applied 24 h later. The adhesion, migration and apoptosis rates, and the cell cycle of the leukemia cells were analyzed after incubation for 24 and 48 h. In contrast to the 2D culture system, the stromal cells in the scaffolds secreted significantly more alkaline phosphatase and Opn (P<0.05). c(RGDfV) disrupted the adhesion and migration between the tumor cells and the matrix, induced the leukemia cells to leave the protective microenvironment and increased their sensitivity to cell cycle-dependent agents (P<0.05). In summary, the data certified that the 3D scaffolds are suitable for the growth of cells, and that c(RGDfV) inhibits the adhesion and migration abilities of leukemia cells in the endosteal niche. Therefore, blocking the function of Opn may be beneficial in the treatment of acute myeloid leukemia.

[The expression and clinical significance of stromal cell-derived factor-1 and CXCR4 in acute leukemia and malignant lymphoma].

OBJECTIVE: To analyze the expression of stromal cell derived factor-1 (SDF-1) and its functional chemokine receptor CXCR4 in patients with acute leukemia and malignant lymphoma. METHODS: CXCR4 expressed on the membrane surface of marrow mononuclear cells was enumerated with 3-color flow cytometry. The serum level of SDF-1 was determined with ELISA assay. RESULTS: The expression of SDF-1 and CXCR4 in acute leukemia for ALL group, the expression of SDF-1 and CXCR4 is (7115.8 +/- 946.5) ng/L and (77.2 +/- 9.7)%; for ANLL group, the expression is (4642.2 +/- 1146.8) ng/L, (38.9 +/- 11.0)% and malignant lymphoma for HD group, the expression of SDF-1 and CXCR4 is (3728.9 +/- 690.9) ng/L and (9.2 +/- 2.7)%; for NHL group, the expression is (4442.1 +/- 1073.0) ng/L and (8.5 +/- 2.4)% patients were higher than that in controls the expression of SDF-1 and CXCR4 is (2369.3 +/- 966.5) ng/L and (2.7 +/- 1.5)% (P < 0.01). In the leukemia group, the patients with extra-marrow infiltration have higher expression of SDF-1/CXCR4 than those without and the patients; in the lymphoma group. CONCLUSIONS: The high expression of SDF-1 and CXCR4 is somewhat correlated with the pathogenetic process and infiltration characteristics of acute leukemia and malignant lymphoma. This can provide a new method to cure the above-mentioned hematologic tumors.

Are there any new insights for G-CSF and/or AMD3100 in chemotherapy of haematological malignants?

AML is a common life-threatening blood system malignancy. The treatment of AML continues to face greater challenges. An abnormal haematopoietic niche with high adhesion and proliferation might be the root cause of resistance and relapse. Most leukaemia cells are stored in the endosteal niche and recess in the G0 phase, and they are not sensitive to varieties of radiotherapies and chemotherapies. G-CSF and AMD3100 are increasingly used in priming chemotherapy. G-CSF can promote leukaemia cells to the cell cycle, which improves the complete remission rate of leukaemia patients. AMD3100, the novel CXCR4 antagonist, could also potentially promote leukaemia cells to cell cycle and improve the susceptibility of leukaemia cells to chemotherapeutic agents. The combination of them enhances anti-leukaemia effect. So in this review, we explore the function of G-CSF and/or AMD3100 in the priming chemotherapy of haematological malignants.

Extramedullary T-lymphoblastic blast crisis in chronic myelogenous leukemia: A case report of successful diagnosis and treatment.

Extramedullary T-lymphoblastic blast crisis of chronic myelogenous leukemia (CML) is uncommon and the prognosis is poor. It was usually misdiagnosed as the co-existence of T-lymphoblastic lymphoma (T-LBL) and CML. In the present study, we report a patient with CML, who developed extramedullary T-lymphoblastic blast crisis and was successfully treated with human leukocyte antigen (HLA)-mismatched stem cell transplantation. The patient was a 44-year-old man who presented with lymphadenectasis and leucocytosis prior to diagnosis. The bone marrow smear, biopsy and fluorescence in situ hybridization (FISH) of Breakpoint Cluster Region/ Abelson murine leukaemia (BCR/ABL) supported the diagnosis of CML in the chronic phase, while the immunohistochemistry of lymph nodes supported the diagnosis of T-LBL. The FISH test for BCR/ABL in lymph node blast cells was performed and the result was positive; therefore, the patient was diagnosed with extramedullary T-lymphoblastic blast crisis of CML. After several courses of combined chemotherapy, the patient was treated with HLA-mismatched stem cell transplantation and obtained continuous remission for 51 months until the present (September 2013).

Features and clinical outcomes in 40 patients with mixed-lineage acute leukemia in a single center.

Mixed-lineage acute leukemia (MAL) is characterized as acute leukemia involving acute myeloid cells and lymphoid cells at the same time. It is easily misdiagnosed because of the dual characteristics involving both lymphoid and myeloid cells and has a poor prognosis. We retrospectively analyzed the features and treatment effectiveness in a single center in 40 patients with MAL. The morphology was consistent with acute lymphoblastic leukemia (ALL) (47.5%) or acute myeloid leukemia (AML) (20%) or was inconclusive (32.5%). Twenty-two patients were characterized as B/myeloid, and 18 patients as T/myeloid. Cytogenetics showed t(9;22)/(Ph(+)) (12.5%) and 11q23/MLL rearrangements (6.25%). The rate of first complete remission for patients undergoing chemotherapy based on the features of both ALL and AML and of either ALL or AML was 71.4 and 42.9%, respectively. The 1-year overall survival rates were 37.5 and 60.0% for chemotherapy and chemotherapy followed by haploidentical hematopoietic stem cell transplantation (HSCT), respectively. The 1-year disease-free survival rates were 25.0 and 50.0% for chemotherapy and chemotherapy followed by HSCT, respectively. These results showed that MAL is confirmed to be a poor-risk disease. The chemotherapy for remission induction should be based on both myeloid cells and lymphoid cells. Transplantation should be performed after the first remission.

Human bone marrow mesenchymal stem cells expressing SDF-1 promote hematopoietic stem cell function of human mobilised peripheral blood CD34+ cells in vivo and in vitro.

PURPOSE: There is mounting evidence demonstrating that stromal cell derived factor-1 (SDF-1) plays an important role in homing of hematopoietic progenitor cells to bone marrow. This study was aimed to assess whether bone marrow mesenchymal stem cells overexpressing exogenous SDF-1 could synergistically promote the homing of CD34(+) (Cluster of Differentiation [CD]) cells to bone marrow of lethally irradiated severe combined immunodeficiency (SCID) mice. METHODS: Human SDF-1 complementary Deoxyribonucleic acid (cDNA) was transfected into bone marrow-derived mesenchymal stem cells with recombinant lentiviral vector. The expression of SDF-1 was detected by real-time Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA), and the ex vivo chemotaxis function on CD34(+) cells was measured by coculture system and Transwell system. SDF-1 gene-modified mesenchymal stem cell (MSC) and CD34(+) cells were infused into lethally irradiated SCID mice and the hematopoietic reconstitution in the recipient mice was examined. RESULTS: Messenger ribonucleic acid (mRNA) and protein of SDF-1 in infected MSC were significantly higher than that of the non-infected control MSC (p < 0.05). The infected MSC have significant chemotaxis effect on CD34(+) cells in vitro and promote hematopoietic reconstitution after CD34(+) cell transplantation in vivo. CONCLUSION: MSC with high-level expression of SDF-1 can synergistically promote hematopoietic reconstitution after CD34(+) cell transplantation in lethally irradiated SCID mice.

[Intracerebral infiltration by monoclonal plasmacytoid cells in Waldenstrom's macroglobulinemia: a case report and review of the literature].

OBJECTIVE: To sum up the clinical experience of the diagnosis and treatment of intracerebral infiltration by monoclonal plasmacytoid cells in Waldenstrom's macroglobulinemia(Bing-Neel syndrome). METHODS: The clinical data of the diagnosis and treatment of a case of Bing-Neel syndrome was analyzed. RESULTS: A 56-year-old male was diagnosed as Waldenstrom's macroglobulinemia one year ago, and presented with persistent headache during the treatment period. Magnetic resonance imaging showed a high intensity area on T2-weighed images in the right frontal lobe which was well enhanced by gadolinium-diethylenetriaminepenta-acetic acid. Infiltration of neoplastic cells was confirmed by biopsy. Immunohistochemical examination showed that mature plasmacytoid cells in the cerebral parenchyma were immunoglobulin M positive. CONCLUSION: Infiltration in CNS (Bing-Neel syndrome) is uncommon in Waldenstrom's macroglobulinemia. As there is no effective therapy for this Bing-Neel syndrome, combination of radiation and chemotherapy should be considered for this situation.

[Effect of the NHE-1-specific inhibitor DMA on pHi, proliferation and apoptosis of HL-60/ADM cells in vitro].

The aim of this study was to evaluate the effect of dimethyl amiloride (DMA), a specific inhibitor of Na(+)/H(+) exchanger-1 (NHE-1), on intracellular pH value (pHi), proliferation and apoptosis of HL-60/ADM cells in vitro. After treatment with DMA at different doses, pHi of HL-60 and HL-60/ADM cell lines were determined by using pH-sensitive fluorescence dye BECEF-AM; the rate of growth inhibition of cells was detected with MTT assay; cell cycle was detected by flow cytometric DNA analysis; cell apoptosis was observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The results showed that pHi in HL-60/ADM cells was higher than that in HL-60 cells. After treatment with DMA at different doses, pHi decreased, the rate of growth inhibition and the rate of apoptotic cells in HL-60/ADM cells were all higher than those in HL-60 cells. Meanwhile, after treatment with DMA during 100 micromol/L to 150 micromol/L, the increase amplitude of G(0)/G(1) phase cells and the decrease amplitude of S + G(2)/M cells in HL-60/ADM cells were higher than those in HL-60 cells. It is concluded that by causing intracellular acidification, the NHE-1-specific inhibitor DMA inhibits proliferation of HL-60/ADM cells and induces apoptosis of HL-60/ADM cells, and the degree of this growth inhibition of HL-60/ADM cells is higher than that of HL-60 cells.

[In vitro effect of all-trans retinoic acid on cell adhesion molecule expression and adhesion capacity of bone marrow stromal cells in patients received peripheral blood stem cell transplantation].

The aim of this study was to evaluate the effect of all-trans retinoic acid (ATRA) on cell adhesion molecule expression and adhesion capacity of bone marrow stromal cells (BMSC) in patients after conditioning treatment for peripheral blood stem cell transplantation (PBSCT). BMSC of 27 patients before and after conditioning treatment for PBSCT were cultured in vitro. After treated with ATRA at 0.01, 0.1, or 1 micromol/L, expression of intercellular adhesion molecule-1 (ICAM-1) protein and vascular adhesion molecule-1 (VCAM-1) protein were detected by flow cytometry, and soluble ICAM-1 (sICAM-1) protein was determined by using radioimmunoassay. Then BMSC was co-cultured with CD34+ cells, and adhesion rate of BMSC to CD34+ cells was measured. The results showed that after pretreatment with conditioning regimen for PBSCT, the expressions of ICAM-1 and VCAM-1 proteins in BMSC and the expression level of sICAM-1 protein in supernatant of BMSC culture were down-regulated, and the adhesion rate of BMSC to CD34+ cells was decreased, after administration of ATRA, the expression of ICAM-1 protein in BMSC, sICAM-1 protein in culture medium and adhesion rate of BMSC to CD34+ cells all increased significantly, but expression of VCAM-1 protein changed no significantly. It is concluded that the ATRA can partly restore adhesion function of BMSC injured by pretreatment for PBSCT and contribute to hematopoietic reconstitution.

[Expression of stromal cell derived factor-1(SDF-1) and its receptor CXCR4 in hematologic malignancies].

The aim was to analyze the expression level of stromal cell derived factor-1 (SDF-1) and its functional chemokine receptor CXCR4 in the patients with hematologic malignant tumor and their clinic significance. 28 patients with hematologic malignant tumor and 12 normal controls were chosen to be experimental objects. CXCR4 expressed on the cell membrane in bone marrow was enumerated by flow cytometry and serum level of SDF-1 was determined by ELISA assay. The result showed that the expression of SDF-1 and CXCR4 in hematologic malignant tumor were higher than that in normal controls, and the expression levels of two molecules were correlated. What is more, the different hematologic malignant tumor had different CXCR4 expression. In conclusion, the high expression of SDF-1 and CXCR4 in serum and bone marrow cells can be used as detective factors to hematologic malignant tumor. A correlation exists between the high expression of CXCR4 and the infiltration of hematologic malignant cells.

[Effects of inhibiting SDF-1 expression by RNA interference on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells].

OBJECTIVE: To observe the effects of inhibiting stromal cell derived factor-1 (SDF-1) expression by RNA interference (RNAi) on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells. METHODS: SDF-1 specific short hairpin RNA (shRNA) expressing plasmid was transferred into cultured human acute leukemic bone marrow stromal cells, positive clones were isolated by screening G418 resistance (Group A) , SDF-1 protein level in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The adhesion rates to bone marrow stromal cells layer and the drug sensitivity to doxorubicin of co-cultured Jurkat cells were detected by cell counting and MTT assay, respectively. The un-transfected bone marrow stromal cells of acute leukemia patient (Group B) or normal subject (Group C) were taken as control. RESULTS: The level of secreted SDF-1 protein (pg/10(5) cells/week) in the supernatants of Group A, B and C were 1920 +/- 205, 12,370 +/- 1355 and 6620 +/- 770, respectively. Of co-cultured Jurkat cells in Group A, B and C, the adhesion rates after 24 h co-culturing were (28.8 +/- 2.6)%, (57.4 +/- 3.8)% and (45.2 +/- 4.0)%, respectively, and the IC50 values of doxorubicin were 585, 6162 and 1758 nmol/L, respectively. CONCLUSION: Down-regulating SDF-1 expression of bone marrow stromal cells by RNAi reduces adhesion rates and enhances drug sensitivity to doxorubicin of their co-cultured Jurkat cells.

[Effects of RNA interference inhibiting SDF-1 expression in bone marrow stromal cells on the proliferation and apoptosis of co-cultured Jurkat cells].

OBJECTIVE: To study the effects of RNA interference inhibiting stromal cell derived factor-1 (SDF-1) expression on the proliferation and apoptosis of co-cultured Jurkat cells. METHOD: Inhibition of SDF-1 expression by RNA interference (RNAi) was achieved by transferring SDF-1 specific short hairpin RNA (shRNA) expressing plasmid into cultured human acute leukemic bone marrow stromal cells. Resistant clones were obtained by G418 selection (group A). The concentration of SDF-1 protein in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The population double time (PDT), cell cycles, apoptosis rates and the expressions of PCNA, Bcl-2/Bax, Fas/FasL of co-cultured Jurkat cells were detected by cells counting, flow cytometry. TdT-mediated dUTP nick-end labelling (TUNEL) and immunocytochemistry (ICC), respectively. The un-transfected acute leukemic (group B) and normal (group C) bone marrow stromal cells were taken as controls. RESULTS: The content of SDF-1 protein in supernatant of group A\[(384 +/- 41) pg/ml] was significantly lower than that in group B[(2474 +/- 271) pg/ml] or group C[(1324 +/- 154) pg/ml]. As group A compared with group B and group C, the PDT of co-cultured Jurkat cells was prolonged (group A: 42 h, vs group B: 29 h, group C: 33 h), and G(0)/G(1) stage cells increased [group A: (28.47 +/- 2.39)%, vs group B: (19.43 +/- 2.80)%, group C: (27.15 +/- 2.07)%], S stage cells decreased [group A: (25.57 +/- 1.90)%, vs group B: (74.48 +/- 3.23)%, group C: (60.99 +/- 2.33)%], G(2)/M stage cells increased [group A: (45.96 +/- 3.24)%, vs group B: (6.09 +/- 1.96)%, group C: (11.86 +/- 1.98)%], the apoptosis rate increased [group A: (15.2 +/- 0.8)%, vs group B: (5.4 +/- 0.7)%, group C: (9.5 +/- 0.4)%], and the expressions of PCNA, Bcl-2, Fas decreased; whereas the expressions of Bax and FasL were increased. CONCLUSION: The inhibition of SDF-1 expression in bone marrow stromal cells inhibits the proliferation and promotes the apoptosis of co-cultured Jurkat cells.

[Influence of anti-CXCR4 monoclonal antibody 12G5 on killing effect of cytosine arabinoside to HL-60 cell].

This study was aimed to explore the influence of anti-CXCR4 monoclonal antibody 12G5 on killing effect of cytosine arabinoside (Ara C) to HL-60 cell, and to assess its therapeutic value in marrow residual disease. HL-60 cells were cultured and co-cultured with leukemic stromal cells, and SDF-1 activity was inhibited with 10 microg/ml 12G5, then, killing effects of Ara C on HL-60 cells were investigated by MTT and morphology assay. Curves by MTT assay revealed that in the test group of 20 microg/ml Ara C, A(540) values decreased slowly but straightly, however, in control group A(540) values decreased markedly for the first two days, and increased from day 3 or 4. In the test group of 40 microg/ml Ara C, although increasing at constricted range of 7 - 9 days, A(540) values decreased in whole observing period of 12 days, while in control group A(540) values decreased markedly at day 0-3, and increased from day 4. Furthermore, two curves go across each other at day 5, and continue the increasing tendency. Morphology results showed that in both treated groups, the number of HL-60 cell decreased markedly and increased gradually in control group, but just contrary to test group. It is concluded that 12G5 may weaken the killing effect of Ara C on HL60 cell in earlier period, but reinforce the total killing effect and delay the occurrence of drug resistance simultaneously. Thus 12G5 has the therapeutic potential on marrow residual disease.

[Effects of anti- CXCR4 monoclonal antibody on adhesiveness of human acute myelocytic leukemia cell line HL-60 and expression of Bcl-2, Fas proteins].

To study the importance of chemokine SDF-1 in surviving of acute myelocytic leukemia cells HL-60, the adhesion ability of HL-60 and expression of Bcl-2, Fas protein when SDF-1 activity was blocked by anti-CXCR4 monoclonal antibody (12G5) were compared with those detected before MAb incubation, in this experiment, HL-60 cell were cultured and co-cultured with normal marrow stromal cell. The adhesion rate was detected while the expression of Bcl-2 and Fas proteins were assayed by immunohistochemical technique when SDF-1 activity was inhibited. The results showed that cell adhesion rate of HL-60 decreased while the expression Bcl-2 decreased and Fas increased. It is concluded that inhibition of SDF-1 activity increases cell apoptosis and thus reduces life-span of leukemia cell at certain level.