Given the tremendous need for and potential of umbilical cord blood (CB) to be utilized as a donor source for hematopoietic stem cell (HSC) transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited HSC and hematopoietic progenitor cell (HPC) numbers available for HSC transplantation from a single collection. We have previously described the use of CD26 inhibitor treatment of donor cells as a method to increase the transplant efficiency of mouse HSCs and HPCs into a mouse recipient. To study the use of CD26 inhibitors as a method of improving the transplantation of human CB HSCs and HPCs, we utilized the nonobese diabetic/severe combined immunodeficient/beta 2 microglobulin null (NOD/SCID/B2m(null)) immunodeficient mouse model of HSC transplantation. We report here significant improvements in the engraftment of long-term repopulating cells following the treatment of either CD34(+) or lineage negative (lin()) donor CB with the CD26 inhibitor, Diprotin A, prior to transplant. These results establish a basis on which to propose the use of CD26 inhibitor treatment of donor CB units prior to transplantation for the purpose of improving transplant efficiency and subsequently patient outcome.
In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.
The chemokine CXCL12 (stromal cell derived factor-1/SDF-1) stimulates hematopoietic stem and progenitor cells (HSCs/HPCs) through the corresponding chemokine receptor CXCR4. CXCL12 is thought to be important for both proper HSC homing, retention, and engraftment into the bone marrow (BM) and mobilization out of the BM. Previous studies suggest that breaking the CXCL12-CXCR4 interaction mobilizes HPCs, blocking CXCR4 inhibits HSC homing, and overexpression increases HSC/HPC repopulation. The efficiency of mobilization and engraftment therefore appears to be dependent on the response of HSCs/HPCs to CXCL12, which is in turn dependent upon levels of CXCR4 expressed on HSCs/HPCs. However, expression of CXCR4 on the surface of HSCs/HPCs appears to be variable. To study the function of CXCR4 on HSCs/HPCs, we used the MSCV-based bicistronic (EGFP) retroviral vector MIEG3 to overexpress CXCR4 on M07e cells, an established model of human HPC. CXCR4 overexpression resulted in significant increases in CXCL12-induced chemotaxis and cell survival. Most importantly, cells overexpressing CXCR4 responded to CXCL12 at levels typically too low induce a response. These data suggest that an increased transplant efficiency resulting from CXCR4 overexpression is likely a function of increased HSC/HPC homing and increased HSC/HPC survival in the recipient's BM. These experiments also validate the ability of the MIEG3-CXCR4 retroviral construct to overexpress CXCR4 efficiently and the use of MIEG3-CXCR4 M07e cells for further study. Finally, this information may have future potential therapeutic implications for improvements in transplant efficiency.
Given the tremendous need for and potential of umbilical cord blood to be utilized as a donor source for hematopoietic stem cell transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited hematopoietic stem and progenitor cell numbers available for hematopoietic stem cell transplantation from a single collection. We have previously described the use of CD26 inhibitor treatment of donor cells as a method to increase the transplant efficiency of mouse hematopoietic stem and progenitor cells into a mouse recipient (Christopherson, KW 2nd, et al, Science2004. 305:1000–1003). We therefore hypothesized that inhibition of CD26 on human donor cord blood prior to transplant would result in an increase in long-term engraftment potential. To test this hypothesis, we isolated CD34 enriched (CD34+) or lineage depleted (lin−) pooled cord blood cells, evaluated their level of CD26 expression and activity, and then tested their ability to engraft into the NOD/SCID/B2mnull immunodeficient mouse model of hematopoietic stem cell transplantation with or without prior CD26 inhibitor treatment. We observed that long-term engraftment into the recipient mouse bone marrow at twelve weeks post sub-lethal irradiation (350cGy), followed by transplantation of 1x105 pooled donor cells was 13.4±2.0% and 55.5±4.0% CD45+ human cells in the untreated, and CD26 inhibitor (Diprotin A) treated CD34+ donor cells respectively. We also observed engraftment levels in the mouse recipient bone marrow of 6.2±0.7% and 47.0±2.8% CD45+ human cells for untreated and CD26 inhibitor treated lin− cells respectively. These measurements represent approximately a 4-fold and 7.5-fold improvement in the engraftment of long-term repopulating cells resulting from CD26 inhibitor treatment of either CD34+ or lin− donor cells respectively. These pre-clinical results establish a basis on which to propose the use of CD26 inhibitor treatment of donor cord blood units prior to transplantation for the purpose of improving transplant efficiency and subsequently patient outcome.
Hematopoietic stem cell transplantation (HSCT) serves as a successful treatment option for patients with malignant or non-malignant severe hematologic diseases. However, large numbers of transplantable donor cells are needed and patient survival is compromised when donor cell numbers are limited, as is the case when umbilical cord blood (CB) donor cells are utilized as a donor source for transplantation into adult patients. Given that CB is readily available, has a lower histocompatibility requirement, and has a reduced risk of graft vs. host disease (GVHD) there are advantages to utilizing CB for HSCT, in particular when an appropriate matched donor is not available. However, in the majority of cases potential recipients have been confined to children because in adults, the amount of CB collected appears to be a limiting factor. Given the promise of CB for HSCT there is significant potential and application for improvements in the efficiency of Hematopoietic Stem Cell (HSC) trafficking to the bone marrow (BM) in both scenarios. To facilitate our long term goal of improving HSCT efficiency, we have recently delineated a novel method by where the inhibition of peptidase CD26 on donor HSC and Progenitor Cells (HPC) increases the number of donor HSC/HPC trafficking to recipient’s BM, resulting in a significant increase in HSC transplant efficiency. CD26 (DPPIV/dipeptidylpeptidase IV) is a membrane bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. Natural substrates of CD26 protease activity include the pancreatic polypeptide family, the glucagon family, and the chemokine family. Since the removal of N-terminal amino acids from chemokines frequently result in significant changes in functional activity, proteolytic cleavage of chemokines has significant implications with respect to their ability to act on cells. We have previously shown that that suppression of CD26 activity, by the use of CD26 inhibitors or the use of CD26−/− donor cells, increases the efficiency of HSC/HPC settlement and growth in the transplanted recipient. This translates into an increase in overall recipient survival in mice. We report here the results of experiments that examine the ability of several cytokines to alter CD26 on the surface of CD34+CD38− phenotypically defined HSC/HPC from CB. HSC isolated from CB were treated with varying dilutions of either Stem Cell Factor (SCF/steel factor/kit ligand), Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), Erythropoietin (EPO), or Trombopoietin (TPO) during culture in IMDM, 20% FBS, 37°C, 5%CO2 for 18 hours and then analyzed for changes in CD26. We observed significant dose dependent increases in the percentage of cells expressing CD26 and the amount of CD26 being expressed on each cell in samples treated with G-CSF, GM-CSF, and SCF as measured by multi-varient flow cytometric analysis. More modest changes in CD26 expression were observed during EPO and TPO treatment. Differential expression of CXCR4 was also noted. We feel these observations have important clinical implications with respect treatment of donor cells prior to transplantation and treatment of patients post HSCT.
The chemokine CXCL12 (stromal cell-derived factor 1/SDF-1) induces the migration of hematopoietic stem and progenitor cells (HSC/HPC) through the corresponding chemokine receptor, CXCR4. CXCL12 is thought to be important for proper homing and engraftment of HSC/HPC to the bone marrow (BM) and mobilization of HSC/HPC out of the BM. Previous studies have suggested that breaking the CXCL12-CXCR4 interaction mobilizes both human and mouse HPC (Liles WC, et al. Blood. 2003 102(8): 2728-30.) (Broxmeyer HE, et al. ASH 44th Annual Meeting. 2002 #2397). Other studies suggest that blocking CXCR4 inhibits homing (Peled A, et al. Science. 1999 283(5403): 845-8) and CXCR4 overexpression by lentiviral vector increases repopulation (Kahn J, et al. Blood. 2004 103(8): 2942-9) of CD34+ cells to the BM of NOD/SCID recipient mice. Expression of CXCR4 on the surface of HSC/HPC appears to be variable, depending highly on the cytokine and growth factor composition of the environment. This may partially explain the reduction in homing efficiency observed during the expansion of cord blood (CB). The efficiency of engraftment therefore appears to be dependent on the response of HSC/HPC to CXCL12 which is in turn dependent upon levels of CXCR4 expressed on HSC/HPC. In order to study the functional cellular response of HSC/HPC independent of variable levels of CXCR4 expression on the surface of cells we utilized the MSCV-based bicistronic (EGFP) retroviral vector, MIEG3, to overexpress human CXCR4 in M07e cells. The human megakaryocytic leukemia cell line, M07e, (a CD34+, c-Kit+, growth-factor-dependent human cell line) has been established as a model for human HPC. Cells infected with the MIEG3 empty vector or the MIEG3-CXCR4 construct were sorted based on their expression of GFP and CXCR4 expression was measured by flow cytometric analysis. MIEG3-CXCR4 M07e cells (GFP+) and MIEG3-CXCR4 bright M07e cells (GFP+++) express significantly higher levels of CXCR4 than M07e cells, MIEG3 M07e cells (GFP+), or MIEG3 bright M07e cells (GFP+++). Migratory response of cells to CXCL12 was assessed by chemotaxis assay. Increased CXCL12 induced chemotaxis was observed in MIEG3-CXCR4 M07e cells (p<0.05) and MIEG3-CXCR4 bright M07e cells (p<0.01). Overexpression of CXCR4 also resulted in a significant increase in CXCL12 induced cell survival during growth factor withdrawal, most markedly at low doses of CXCL12 (10 and 1.0ng/ml) (p<0.05). Thus, retroviral overexpression of CXCR4 enhances both the migratory response and survival of the human HPC line, M07e, to CXCL12. Most importantly cells respond to levels of CXCL12 that are normally too low. This data validates the ability of the MIEG3-CXCR4 construct to efficiently overexpress CXCR4 and validates the future use of MIEG3-CXCR4 M07e cells for further study of CXCR4 in HPC. This is important given the previously established ability of MIEG3 to infect CD34+ CB cells (Tao W et al. Gene Ther. 2004 11(1): 61-9). This information also suggests that previously reported increases in engraftment resulting from CXCR4 overexpression is a function of both increased HSC/HPC settlement and increased HSC/HPC survival in the transplant recipient’s BM. This information may have potential therapeutic application for improvements in overall transplant efficiency.
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