ABSTRACT
Chronic myeloid leukemia (CML) is a malignant clonal disease derived from hematopoietic stem cells. CML stem cells were thought to be the root which could lead disease development and ultimately rapid change. However, a stable animal model for studying the characteristics of CML stem cells is currently lacking. This study was aimed to establish a transplanted human CML nude-mice model to further explore the biological behavior of CML stem cells in vivo, and to enrich CML stem cells in nude mice by series transplantation. The 4 - 6 weeks old BALB/c nude mice pretreated by splenectomy (S), cytoxan intraperitoneal injection (C) and sublethal irradiation (I) were transplanted intravenously with (5 - 7) × 10(7) of bone marrow mononuclear cells from CML patients in chronic phase. Alternatively, 4 - 6 weeks old BALB/c nude mice pretreated by lethal irradiation were transplanted intravenously with 5 × 10(6) homologous bone marrow cells of BALB/c nude mice together with (5 - 7) × 10(7) of bone marrow mononuclear cells from CML patients in chronic phase simultaneously. The leukemic cells engrafted and infiltrated in organs and bone marrow of the mice were tracked by reverse transcription-polymerase chain reaction (RT-PCR), plastic-embedded biopsy and flow cytometry. The results of these two methods were compared. The results showed that human CML cells engrafted and infiltrating into the bone marrow of two nude mice pretreated with SCI could be detected. In spite of the low successful rate, results suggested the feasibility of this method by using BALB/c nude mice as a human CML animal model. In contrast, in nude mice pretreated by the lethal dose irradiation, CML cells in the bone marrow could not be found. It is concluded that human bone marrow CML cells can results in leukemia in nude mice pretreated by SCI. Thus this study provides a new strategy for establishment of CML animal models which deserves further elaboration.
Subject(s)
Animals , Humans , Male , Mice , Disease Models, Animal , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Mice, Nude , Mice, SCID , Neoplasm Transplantation , Neoplastic Stem Cells , Transplantation, HeterologousABSTRACT
The aim of this study was to investigate the apoptosis-inducing effect of anti-CD44 monoclonal antibody IM7 on chronic myeloid leukemia (CML) stem/progenitor cells in vitro and to explore its possible mechanism. Leukemic stem/progenitor cells (LSPCs) expressing CD34(+), CD38(-) and CD123(+) were isolated from bone marrow (BM) cells of 20 patients with newly-diagnosed chronic myeloid leukemia by using EasySep(TM) magnetic beads. The percentage of apoptotic CML-LSPCs was assayed by Annexin-V/PI staining; the expression changes of c-myc and NF-kappaB mRNA were detected by real-time quantitative PCR (RQ-PCR) and RT-PCR; the NF-kappaB activity was detected by NF-kappaB Activation Nuclear Translocation Assay Kit; the BCL-2 protein expression was determined in the Western blot method. The results showed that the IM7 effectively induced apoptosis of CML-LSPCs; the mean percentage of early apoptotic cells significantly increased, as compared with the untreated control CML-LSPCs cells 12.58 +/- 2.84% vs 5.42 +/- 1.84% (p < 0.05). The c-myc, NF-kappaB mRNA expressions were down-regulated as compared with the control group (0.65 +/- 0.10 vs 1.00, 0.42 +/- 0.21 vs 1.00, respectively) (p < 0.01) by RQ-PCR and (0.49 +/- 0.09 vs 0.60 +/- 0.12, 0.47 +/- 0.11 vs 0.67 +/- 0.08, respectively)(p < 0.01) by RT-PCR. The BCL-2 protein level in CML-LSPCs treated with IM7 also decreased as compared with the control group (p < 0.01). In addition, the depression of NF-kappaB activity was observed through fluorescence microscope. It is concluded that the anti-CD44 monoclonal antibody IM7 effectively induces apoptosis of CML-LSPCs through down-regulating c-myc and bcl-2 mRNA expression, and decreasing NF-kappaB activity in CML-LSPCs.
Subject(s)
Humans , Antibodies, Monoclonal , Pharmacology , Apoptosis , Genetics , Hyaluronan Receptors , Allergy and Immunology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Genetics , Metabolism , NF-kappa B , Metabolism , Neoplastic Stem Cells , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Proto-Oncogene Proteins c-myc , Metabolism , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell (CML-LSC) and its mechanism.</p><p><b>METHODS</b>CD34(+)CD38(-)CD123(+) leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia (CML) patients BM cells and CD34(+)CD38(-) hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySep(TM) magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM), and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay, respectively.</p><p><b>RESULTS</b>(1) After incubated with IM7, the LSC and HSC CD44 expression rates were (86.60 ± 2.10)% vs. (25.40 ± 1.70)% (P < 0.05), respectively. (2) The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7, the OD value (A(570)) changing from pre-incubation of (0.62 ± 0.11) to post-incubation of (0.34 ± 0.07), while there was little change of A(570) in the HSC group. (3) The migration ability of the LSC group was inhibited evidently after incubated with IM7, the inhibition rate being 46% ∼ 63%, while little change of that in HSC group was detected. (4) The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7, while little change was found in that of HSC group.</p><p><b>CONCLUSION</b>The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro, which might provide a theoretical evidence for targeting therapy.</p>