MMP-14 Mediates Migration of Acute Myelogenous Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2943-2943
Author(s):  
Neeta Shirvaikar ◽  
Ali Jalili ◽  
Imran Mirza ◽  
Sara Ilnitsky ◽  
Chris Korol ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Myeloid Cell ◽  
Myelogenous Leukemia ◽  
Migration And Invasion ◽  
Rt Pcr ◽  
Multiple Tumor ◽  
Matrigel Invasion ◽  
Tnf Α ◽  
Acute Myelogenous

Abstract Matrix metalloproteinase (MMP)-14 expression correlates with progression and metastasis of multiple tumor cell types and is a major mediator of cell migration and invasion. MMP-14 possesses a trans-membrane domain that tethers the enzyme to the plasma membrane and not only activates proMMP-2 but also degrades extracellular matrix (ECM) by pericellular proteolysis and cleaves several non-ECM molecules, including adhesion molecules and chemokines. To assess the role of MMP-14 in leukemic dissemination we evaluated its expression in myeloid cell lines and primary acute myelogenous leukemic (AML) samples. Using RT-PCR, flow cytometry and Western blotting we found that MMP-14 is highly expressed in leukemic myeloid cell lines THP-1, U937, HEL and K562; and primary samples from 37 out of 40 patients (pts) diagnosed with AML (WHO classification, AML with recurrent cytogenetic translocations: 9 pts; AML with multilineage dysplasia: 4 pts; AML not otherwise categorized: 27 pts). Moreover, primary AML blasts secreted the 72 kDa proenzyme form of MMP-2 into media (zymography) which became activated after co-culture of AML blasts with bone marrow (BM) stromal cells. This activation was inhibited by the potent MMP-14 inhibitor epigallocatechin-3-gallate (EGCG). We also found that migration of primary AML cells (trans-Matrigel invasion assay) was inhibited by EGCG; and silencing of MMP-14 by transfecting THP-1 cells and AML blasts with MMP-14 siRNA oligonucleotides resulted in reduced trans-Matrigel migration of these cells. Given that AML blasts constitutively secrete TNF-α, we confirmed that AML blasts express mRNA for TNF-α as well as its receptors TNFR1 and TNFR2; and TNF-α levels are elevated in the plasma of AML patients. However, we demonstrated that recombinant human (rh) TNF-α further upregulated MMP-14 expression in AML blasts (RT-PCR, Western blot) and increased its incorporation into membrane lipid rafts (confocal microscopy). Moreover, rh TNF-α- stimulated AML blasts were found to be more potent activators of pro-MMP-2 than unstimulated cells (zymography), indicating that activation may occur via upregulation of MMP-14; had significantly higher migration which was inhibited by EGCG and also by the TNF-α receptor inhibitor Enbrel; and rh TNF-α had no effect on the migration of MMP-14 siRNA-silenced AML cells. Other factors tested, for example, SDF-1, IL-1 and TGF-β, had no effect on MMP-14 expression in AML cells (flow cytometry). In conclusion, we report here for the first time that MMP-14 is expressed in AML blasts and is modulated by TNF-α. We suggest that, by pericellular degradation of ECM and activation of latent MMP-2 secreted by AML blasts, MMP-14 may contribute to the highly proteolytic BM microenvironment in AML and the invasive phenotype of this malignancy.

Membrane Type-Matrix Metalloproteinases (MT-MMPs) Are Expressed in Acute Myeloid Leukemia Cells and Are Upregulated by TNF-α.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2853-2853
Author(s):  
Neeta Shirvaikar ◽  
Ali Jalili ◽  
Sara Ilnitsky ◽  
Loree Larratt ◽  
Imran Mirza ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Cell Lines ◽  
Western Blotting ◽  
Lymphoid Cell ◽  
Myeloid Cell ◽  
Rt Pcr ◽  
Cd34 Cells ◽  
Tnf Α ◽  
Membrane Type ◽  
Lymphoid Cell Lines

Abstract Membrane type-matrix metalloproteinases (MT-MMPs) play a key role in tumor cell progression and metastasis but their role in hematological malignancy and leukemic cell dissemination is still unclear. To date, six MT-MMPs have been identified, of which four (MT1-, MT2-, MT3- and MT5-MMP) possess a trans-membrane domain that tethers the enzyme to the plasma membrane and two (MT4- and MT6-MMP) are anchored to the cell surface via a glycophosphatidyl inositol domain. MT-MMPs are known to activate pro-forms of MMPs. We examined the expression of all six MT-MMPs in 13 myeloid and lymphoid cell lines using RT-PCR, Western blotting and FACS. We found that MT1-MMP was expressed on most of the cell lines tested, MT2-MMP was expressed in myeloid cell lines such as THP-1, HEL, K562 and U937, and in T cell lines such as Jurkat and CEM, but not in any of the tested B cell lines, MT3-MMP was not expressed in any of the cell lines except for THP-1, MT4-MMP was strongly expressed in all myeloid but not lymphoid cell lines and MT5-MMP and MT6-MMP were expressed in most of the myeloid cell lines. Next, we examined the expression of all six MT-MMPs in primary AML samples and found that MT1-MMP is expressed in 20 out of 24 AML patient samples as detected by RT-PCR and Western blotting, MT2-MMP and MT4-MMP were expressed in 21 and 22 out of 24 samples, respectively, and MT6-MMP was expressed in 17 out of the 17 samples tested. In contrast, MT3- and MT5-MMPs were not found in the primary AML samples. Zymographic analysis showed that the pro form of MMP-2 was secreted in media conditioned by AML blasts and became activated only when these AML blasts were co-cultured with BM stromal cells. Since TNF-α is endogenously secreted by AML blasts we next stimulated these cells with human recombinant TNF-α and found strong upregulation of MT1-MMP and MT6-MMP. Moreover, zymography demonstrated that TNF-α-stimulated AML blasts are more potent in activation of pro-MMP-2 than unstimulated cells, indicating that activation may occur via upregulation of MT-MMPs. Furthermore, we found that TNF-α stimulation led to stronger upregulation/induction of MT1- and MT2-MMPs in CD34+ cells than in CD34- cells isolated from AML patients. In conclusion, we report here for the first time that MT1-, 2-, 4- and 6-MMPs are expressed in AML blasts and suggest that these MT-MMPs may contribute to the invasive phenotype of this malignancy.

Latent Infection and Reactivation of Human Herpesvirus 6 in Two Novel Myeloid Cell Lines

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 991-999 ◽  
Author(s):  
Masaki Yasukawa ◽  
Hideki Ohminami ◽  
Eiji Sada ◽  
Yoshihiro Yakushijin ◽  
Masahiko Kaneko ◽  
...  
Keyword(s):  
Cell Lines ◽  
Philadelphia Chromosome ◽  
Human Herpesvirus ◽  
Myeloid Cell ◽  
Antigen Expression ◽  
Cell Lysis ◽  
Myelogenous Leukemia ◽  
Human Herpesvirus 6 ◽  
Latently Infected ◽  
Herpesvirus 6

Abstract It has been reported that reactivation of human herpesvirus-6 (HHV-6) causes a failure of hematopoiesis. To clarify the mechanisms of bone marrow suppression induced by HHV-6 infection, it is necessary to establish an in vitro model of HHV-6 infection in hematopoietic progenitor cells. We have established two novel Philadelphia chromosome–positive myeloid cell lines, SAS413 and SAS527, which possess different hematologic characteristics and show distinct susceptibility to infection by HHV-6, from a patient with blast crisis of chronic myelogenous leukemia (CML). HHV-6 subgroup A (HHV-6A) showed marked replication in SAS413, forming syncytia and inducing cell lysis in short-term culture. On the other hand, HHV-6A–inoculated SAS527 continued to proliferate without cell lysis and only a few cells showed HHV-6 antigen expression. In contrast to HHV-6A infection, inoculation with HHV-6 subgroup B (HHV-6B) did not induce any cytopathic effect (CPE) or viral antigen expression in either of the cell lines. Although HHV-6B replication was undetectable, the presence of the HHV-6 genome in both cell lines was shown by polymerase chain reaction (PCR) during culture for more than 10 months, suggesting that HHV-6B latently infected SAS413 and SAS527. Phorbol ester treatment of SAS527 latently infected with HHV-6B resulted in reactivation of HHV-6, as shown by the appearance of a CPE, positive reactivity for the HHV-6 antigen, and isolation of infectious HHV-6. These novel cell lines should be useful for studying the mechanisms of HHV-6–induced hematopoietic failure and HHV-6 latency and reactivation, as well as differentiation, of the myeloid cell lineage.

Terminal Deoxynucleotidyl Transferase Expression in Acute Myelogenous Leukemia and Myelodysplasia as Determined by Flow Cytometry

2000 ◽  
Vol 37 (3-4) ◽  
pp. 319-331 ◽  
Author(s):  
Yang O. Huh ◽  
Terry L. Smith ◽  
Patricia Collins ◽  
Carlos Bueso-Ramos ◽  
Maher Albitar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myelogenous Leukemia ◽  
Myelogenous Leukemia ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Acute Myelogenous

scholarly journals Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1074-1084 ◽  
Author(s):  
T Nagai ◽  
H Harigae ◽  
H Ishihara ◽  
H Motohashi ◽  
N Minegishi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transcriptional Activation ◽  
Early Stage ◽  
Blot Hybridization ◽  
Myeloid Cell ◽  
Myelogenous Leukemia ◽  
Molecular Heterogeneity ◽  
Human Leukemia ◽  
Gata Factor ◽  
Gata Factors

Abstract To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2- specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.

scholarly journals Heat stress combined with lipopolysaccharide alter the activity and superficial molecules of peripheral monocytes

2019 ◽  
Vol 33 ◽  
pp. 205873841982889
Author(s):  
Jiajing Luo ◽  
Yi Chen ◽  
Chengjia Ding ◽  
Jialing Qiu ◽  
Yulan Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Heat Stress ◽  
Western Blot ◽  
Inflammatory Mediators ◽  
Peripheral Blood ◽  
Heat Stroke ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α

The purpose of this study was to focus on the underlying relationship between the hyperactivity for the peripheral monocytes and heat stroke by investigating the inflammatory oxidative activity of and the expression of superficial molecules. Peripheral blood samples were collected from 10 healthy adult volunteers. Human blood monocytes were isolated by density gradient centrifugation and sequent adherent culture. The objectives were divided into four groups: 43°C heat stress combined with lipopolysaccharide (LPS) group, 43°C heat stress group, LPS group, and control group. There were 10 cases in each group. An enzyme-linked immunosorbent assay (ELISA) test was used to measure the concentrations of supernatant inflammatory mediators (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10)). After loaded by 2,7-Dichlorodi-hydrofluorescein-diacetate (DCFHDA) fluorescent probe, intracellular reactive oxygen species (ROS) levels were determined by a flow cytometry. After fluorescent microspheres incubation, the phagocytosis of monocytes was observed under a fluorescent microscope. Respectively, the flow cytometry and Western blot were used to evaluate the level of triggering receptor expressed on myeloid cells-1 (TREM-1) and Toll-like receptor-4 (TLR-4) on the monocytes. Furthermore, the mRNA expression of TREM-1 and TLR-4 was detected by real-time polymerase chain reaction (RT-PCR). The heat stress combined with LPS stimulation promoted the peripheral monocytes to produce inflammatory mediators (TNF-α, IL-1β, and IL-10) and release ROS. Otherwise, such complex strike significantly suppressed the phagocytic activity of monocytes in peripheral blood. Moreover, the expression of TREM-1, TLR-4 and CD86 was measured by the flow cytometry on peripheral monocytes which were respectively promoted by the union of heat stress and LPS. The results of Western blot and RT-PCR demonstrated the similar kinetics on these superficial molecules (TREM-1, TLR-4, and CD86) stimulated by the combination of heat stress and LPS. The underlying mechanism of the dysfunction for the peripheral monocytes may be related to the abnormal expression of superficial molecules TREM-1, TLR-4, and CD86 on the monocytes induced by heat stress and LPS.

scholarly journals Persistence of multipotent progenitors expressing AML1/ETO transcripts in long-term remission patients with t(8;21) acute myelogenous leukemia

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4789-4796 ◽  
Author(s):  
T Miyamoto ◽  
K Nagafuji ◽  
K Akashi ◽  
M Harada ◽  
T Kyo ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Acute Myelogenous Leukemia ◽  
Fusion Gene ◽  
Phosphoglycerate Kinase ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Differentiation Potential ◽  
Acute Myelogenous

The leukemia-specific AML1/ETO fusion gene has been shown to be detected by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. In the present study, the AML1/ETO mRNA could be detected by RT-PCR in bone marrow (BM) and/or peripheral blood (PB) samples from all 18 patients who had been maintaining complete remission for 12 to 150 months (median, 45 months) following chemotherapy or PB stem cell transplantation (PBSCT), whereas it could not be detected in four patients who had been maintaining remission for more than 30 months following allogeneic BM transplantation (BMT). We surveyed the expression of AML1/ETO mRNA in clonogenic progenitors from BM in these cases. Notably, 51 of 2,469 colonies from clonogenic progenitors (2.1%) expressed the AML1/ETO mRNA in 18 cases who were RT- PCR+ in BM and/or PB samples. Expression was observed in various clonogenic progenitors, including granulocyte-macrophage colonies, mixed colonies, erythroid colonies, and megakaryocyte colonies. Furthermore, we analyzed the clonality of these progenitors by X- chromosome inactivation patterns of the phosphoglycerate kinase (PGK) gene in four female patients. The AML1/ETO mRNA+ progenitors showed the PGK allele identical to that detected in the leukemic blasts from the time of initial diagnosis. Normal constitutive hematopoiesis was sustained by polyclonal BM reconstitution in these patients. Accordingly, these committed progenitor cells that express AML1/ETO mRNA during remission likely have arisen from common t(8;21)+ pluripotent progenitor cells with at least trilineage differentiation potential. These data strongly suggest that the origin of the clonogenic leukemic progenitors of t(8;21) AML may be multipotent hematopoietic progenitors that acquired the t(8;21) chromosomal abnormality.

Microrna Expression Profiling in Acute Myelogenous Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1131-1131
Author(s):  
Fernando J. Suarez Saiz ◽  
Serban San-Marina ◽  
Mark D. Minden
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Acute Myelogenous Leukemia ◽  
Myelogenous Leukemia ◽  
Erythroid Cell ◽  
Normal Bone Marrow ◽  
Hematopoietic Differentiation ◽  
Normal Bone ◽  
Acute Myelogenous

Abstract Acute myelogenous leukemia (AML) arises due to changes in gene expression that block or alter the normal differentiation program of hematopoietic stem cells. A variety of mutations in protein-encoding genes have been shown to contribute to the development of leukemia. Recently a new class of genes called microRNAs (miRNAs) have been identified. miRNAs are a subgroup of highly conserved, non-coding RNAs found only in eukaryotes. They do not encode proteins, and appear to have a significant effect on the proteome of a cell. Their conservation between species suggests their involvement in important biological functions, and in fact been shown to be involved in hematopoietic differentiation. While the function of most miRNAs is still unknown, it is believed that they regulate expression of target mRNAs by using the siRNA machinery either to promote degradation of the mRNA or to block its translation. To begin to understand the role of miRNAs in AML, we used Quantitative Polymerase Chain Reaction (QPCR) to measure the expression level of 20 miRNA precursors in the pro erythroid cell line K562, the pro-myelocytic cell line NB4, the myelomococytic cell line OCI/AML2, AML patients’ blasts and in normal bone marrow (NBM). The investigated miRNAs included some that are known to be specific for hematopoietic tissues or involved in hematopoietic differentiation, as well as all the miRNAs in chromosome 7, a hot spot for gene deletion in AML. Our findings indicate that miRNAs are differentially expressed in patients and cell lines when compared among themselves and against normal bone marrow. For example pre-miR-142 was expressed in NBM and K562 but was found to be elevated in OCI/AML2, NB4 and in all patient samples. Pre-miR-20 was found to be overexpressed in only a subset of patients. Other miRNAs like pre-miR-335 and pre-miR-148a were expressed in NBM and in some patients and not in the cell lines. In an effort to identify possible regulators of miRNA expression, we analyzed the upstream region of pre-miR-142 and found an LMO2 binding site. In AML, the LMO2 gene can be overexpressed relative to normal bone marrow and healthy lymphocytes. This transcription factor is involved in the regulation of genes important in the development of blood cells. To investigate if LMO2 could be involved in the regulation of miR-142 expression, we performed chromatin immunoprecipitation (ChIP) from K562 using an anti-LMO2 antibody. Only the LMO2 immunoprecipitation, and not those from the pre-immune control, were enriched in promoter DNA for pre-miR-142. This is consistent with the observation that miRNAs and coding RNAs can be regulated by the same environmental signals. Based on this observation we propose that oncogenes regulate in part the phenotype and biological behaviour of leukemia by affecting the expression of miRNAs. This further suggests that different forms of leukemia may be recognized based upon the spectrum of miRNAs they express.

Expression of Cyclooxygenase-2 (COX-2) in Human Leukemias and Hematopoietic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4336-4336 ◽  
Author(s):  
Michael B. Lilly ◽  
Leslie Drapiza ◽  
Milan Sheth ◽  
Marina Zemskova ◽  
Svetlana Bashkirova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Growth Factor ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Myelogenous Leukemia ◽  
Human Leukemia ◽  
Flow Cytometry Assay ◽  
Acute Leukemias ◽  
Cox 2 ◽  
Cox 2 Inhibitors

Abstract COX-2 has been implicated in the development of many epithelial cancers, as well as in tumor angiogenesis. COX-2 inhibitors have been shown to have anti-tumor activity in experimental cancer. Little information exists, however, on the expression or role of COX-2 in hematologic malignancies. We have use a variety of immunochemical assays to document expression of COX-2 in human and murine leukemias and hematopoietic cells. The factor-dependent murine cell lines FDCP1 and 32D expressed COX-2 when growing continuously in the presence of IL-3; expression declined markedly when growth factor was removed. FDCP1 cells constitutively expressing bcl-2, pim-1, or bcr-abl had markedly elevated levels of COX-2, and continued to express this enzyme even after removal of growth factor. To assess COX-2 expression in human hematopoietic cells we developed a flow cytometry assay using a FITC-labelled anti-COX-2 MoAb (Cayman). Cells were washed once in serum-free medium, fixed briefly in 1% paraformaldehyde, permeabilized with PBS/0.2% Triton X100, then stained with antibody. Negative control samples were processed similarly but stained with antibody that had been preincubated with immunizing peptide. Specific COX-2 staining was interpreted as the difference between the histograms from blocked versus unblocked anti-COX-2 antibody, as determined by Kolmogorov-Smirnoff analysis. In buffy coat preparations from normal donors, we found constitutive expression of COX-2 in lymphocytes (both B-cells and T-cells). In contrast little or no COX-2 was detected in unstimulated neutrophils or monocytes. In human acute myelogenous leukemia (AML) cell lines we found COX-2 expression to be universal and easily detected. In several cell lines we confirmed the results of our flow cytometry assay with immunoblotting. We further examined 25 cryopreserved samples of human acute leukemia blasts obtained from peripheral blood. COX-2 expression was variable, but universal. Levels generally were less than those seen in immortalized cell lines, and did not correlate with blasts morphology (AML, ALL, APL, AMoL, CML-BT). To determine if COX-2 inhibitors could play a role in the treatment of acute leukemias, we performed cytotoxicity assays using the COX-2 specific inhibitors, celecoxib and NS398. Survival and growth of human AML cell lines were inhibited by both agents. These data demonstrate that 1) a variety of oncogenes can induce expression of COX-2 in hematopoietic cells, 2) clinical human acute leukemias uniformly express COX-2 in circulating blasts, and 3) COX-2 inhibitors are cytotoxic for human leukemia cells. Combination therapies for acute leukemias may evaluate the incorporation of COX-2 inhibitors for added cytotoxic effects or angiogenesis inhibition.

Expression of Leukemia-Associated Antigens (LAA) in Patients with Acute Myelogenous Leukemia (AML).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4497-4497
Author(s):  
Bjoern Schoettker ◽  
Peter Reimer ◽  
Volker Kunzmann ◽  
Hermann Einsele ◽  
Florian Weissinger
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Expression Pattern ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Specific Immunotherapy ◽  
Disease Free Survival ◽  
Myelogenous Leukemia ◽  
Proteinase 3 ◽  
Acute Myelogenous

Abstract Introduction: Immunotherapeutic approaches may be effective additional options to conventional chemotherapy and stem cell transplantation regimens in the treatment of patients with acute myelogenous leukaemia (AML). Without eliciting significant side effects they may help to prolong disease-free survival or at least may delay the time to progression. Immuniotherapeutic strategies comprise vaccination with professional antigen-presenting dendritic cells or transfusion of T-cells with specific anti-leukemic activity. Leukemia-associated antigens are useful targets as they are expressed predominantly in the malignant cells and constitutively not detectable in normal tissue, or as they are at least significantly overexpressed in the leukemic blasts. To determine potential targets for specific immunotherapy in our patients with AML, we started to analyze the antigen profiles of these patients’ leukemia cells using the previously characterized leukemia-associated antigens survivin, WT1, and proteinase-3 (with the HLA-A2-specific nonameric peptide PR-1), and c-Ski, an antigen recently found to be predominantly but not exclusively overexpressed in AML with deletion or monosomy of chromosome 7, indicating a poor prognosis. Methods: The mRNA expression of the above mentioned antigens was examined by conventional RT-PCR. Blasts from patients with AML were collected at diagnosis or at first relapse from peripheral blood and analyzed. Results: The antigen expression pattern in comparison to the AML cell lines HL-60 and K-562 is shown in Table 1. Conclusions: Although treatment of AML patients became more effective during the last decades, high relapse rates contribute to the poor prognosis of the disease. Additional therapeutic strategies are needed to help and prevent disease relapse. Immunotherapy directed to LAA might elicit specific CTL responses effectively eliminating minimal residual disease. Using the LAA profile shown above, we should be able to determine at least one antigen in each patient with AML to serve as a target for individual specific immunotherapy with antigen-loaded autologous dendritic cells or /and specific autologous T-cells. Table 1: Expression pattern of the LAA survivin, WT1, proteinase-3 und c-Ski in AML cell lines HL-60 and K-562, and in seven patients with AML. +/++/+++ positive, - negative, n.d. not determined yet -actinβ survivin WT-1 PRO3 c-ski HL-60 +++ +++ ++ + +++ K-562 +++ ++ ++ − − B.R. +++ +++ ++ +++ +++ B.G. +++ − ++ + − B.C. +++ − n.d. n.d. n.d. E.B. +++ − − n.d. n.d. E.E. +++ +++ +++ ++ + S.T. +++ +++ +++ ++ + V.B. +++ +++ n.d. n.d. n.d.

Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4099-4099
Author(s):  
Zhenhua Qiao ◽  
Xiujuan Zhao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Hematopoietic Cell ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

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