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Jose A Cancelas

Publications and source records attributed to Jose A Cancelas.

12 recordsLinked to original sources
PubMed2007

Stem cell collection and gene transfer in Fanconi anemia.

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34(+) stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34(+) cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34(+) cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

Adolescent↗
PubMed2006

Inhibition of RhoA GTPase activity enhances hematopoietic stem and progenitor cell proliferation and engraftment.

Ras-related Rho GTPases regulate actin cytoskeletal organization, adhesion, gene transcription, and cell-cycle progression. The Rac subfamily of Rho GTPases and Cdc42 has been shown to play essential roles in hematopoietic stem cell (HSC) engraftment and mobilization. Here, we study the role of RhoA, a related Rho GTPase, in HSC functions. Using retrovirus-mediated gene transfer of a dominant-negative (DN) mutant of RhoA (RhoAN19), we demonstrate that down-regulation of RhoA activity resulted in increased HSC engraftment and self-renewal as measured by competitive repopulation and serial transplantation assays. However, overexpression of RhoAN19 resulted in decreased migration toward SDF-1alpha and alpha(4)beta(1)- and alpha(5)beta(2)-integrin-mediated adhesion of hematopoietic progenitor cells in vitro. Low RhoA activity was associated with higher proliferation rate of hematopoietic progenitor cells and increased cells in active phases of cell cycle, most likely via decreasing p21Cip/Waf expression and increasing cyclin D1 levels. Thus, reducing RhoA activity by optimizing the balance between adhesion/migration and proliferation/self-renewal results in a net increase in HSC engraftment. This mechanism could provide a novel therapeutic target to enhance HSC therapies.

Animals↗
PubMed2006

The FIP1L1-PDGFRA fusion gene cooperates with IL-5 to induce murine hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL)-like disease.

Dysregulated tyrosine kinase activity by the Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha (PDGFRA) (F/P) fusion gene has been identified as a cause of clonal hypereosinophilic syndrome (HES), called F/P-positive chronic eosinophilic leukemia (CEL) in humans. However, transplantation of F/P-transduced hematopoietic stem cells/progenitors (F/P(+) HSCs/Ps) into mice results in a chronic myelogenous leukemia-like disease, which does not resemble HES. Because a subgroup of patients with HES show T-cell-dependent interleukin-5 (IL-5) overexpression, we determined if expression of the F/P fusion gene in the presence of transgenic T-cell IL-5 overexpression in mice induces HES-like disease. Mice that received a transplant of CD2-IL-5-transgenic F/P(+) HSC/Ps (IL-5Tg-F/P) developed intense leukocytosis, strikingly high eosinophilia, and eosinophilic infiltration of nonhematopoietic as well as hematopoietic tissues, a phenotype resembling human HES. The disease phenotype was transferable to secondary transplant recipients of a high cell dose, suggesting involvement of a short-term repopulating stem cell or an early myeloid progenitor. Induction of significant eosinophilia was specific for F/P since expression of another fusion oncogene, p210-BCR/ABL, in the presence of IL-5 overexpression was characterized by a significantly lower eosinophilia than IL-5Tg-F/P recipients. These results suggest that F/P is not sufficient to induce a HES/CEL-like disease but requires a second event associated with IL-5 overexpression.

Animals↗
PubMed2006

The role of chemokine activation of Rac GTPases in hematopoietic stem cell marrow homing, retention, and peripheral mobilization.

Signaling downstream from the chemokine receptor CXCR4, the tyrosine kinase receptor c-kit and beta1-integrins has been shown to be crucial in the regulation of migration, homing, and engraftment of hematopoietic stem cells and progenitors. Each of these receptors signal through Rac-type Rho guanosine triphosphatases (GTPases). Rac GTPases play a major role in the organization of the actin cytoskeleton and also in the control of gene expression and the activation of proliferation and survival pathways. Here we review the specific roles of the members of the Rac subfamily of the Rho GTPase family in regulating the intracellular signaling of hematopoietic cells responsible for regulation of homing, marrow retention, and peripheral mobilization.

Animals↗
PubMed2006

In vitro and in vivo evaluation of LEUKOSEP HRC-600-C leukoreduction filtration system for red cells.

BACKGROUND: Documentation of the benefits of leukoreduction has led to the increased use of this technique and the need for development of efficient and effective techniques for its accomplishment. This study investigated the in vitro properties and in vivo autologous radiolabeled recovery of leukoreduced red cells (RBCs) produced through a leukoreduction filtration system for RBCs (LEUKOSEP HRC-600-C, Hemerus Medical). STUDY DESIGN AND METHODS: Normal subjects donated 36 units of RBCs that were leukoreduced on Days 0, 3, or 5 through a "hands-off" technique. Biochemical studies were performed before and after filtration and at the end of 42 days of storage. Units leukoreduced on Days 0 or 5 were held until Day 42 and used for autologous radiolabeled return to determine recovery with 51Cr single-label radiolabeling techniques. RESULTS: Leukoreduction filtration was accomplished in 16.3 +/- 2 minutes on Day 0 at room temperature or 27 to 30 minutes on Days 3 or 5 after refrigeration. Leukoreduction efficiency was 4.6 +/- 0.6 log with a median residual white blood cell (WBC) content of fewer than 3.3 x 10(4) WBCs per unit. RBC recovery was 90 +/- 2 percent. Hemolysis was 0.34 +/- 0.16 percent at the end of 42 days of storage. The in vivo recovery of radiolabeled RBCs 24 hours after autologous return was 80.6 +/- 4.5 percent for RBC units leukoreduced on Days 0 and 5 combined. CONCLUSION: The LEUKOSEP HRC-600-C WBC reduction filtration system produced leukoreduced RBCs efficiently and effectively with acceptable poststorage biochemical measures and posttransfusion recovery after 42 days of storage.

Adult↗
PubMed2006

Children are not little adults: just ask their hematopoietic stem cells.

HSCs differ during ontogeny in some important parameters, including anatomic site of residence and cell cycling characteristics. In this issue of the JCI, Bowie et al. show that postnatal HSCs as well as fetal liver HSCs in mice are active in the cell cycle at much higher rates than that of adult HSCs; however, this increased frequency of cycling abruptly ceases 4 weeks after birth (see the related article beginning on page 2808). The cycling postnatal HSCs expressed high levels of CXC chemokine ligand 12 (CXCL12, also known as stromal cell-derived factor 1 [SDF-1]), a chemokine previously implicated in stem cell trafficking to the marrow cavity and shown to be expressed by cells within the hematopoietic microenvironment. These cells also possessed an engraftment defect impeding reconstitution in irradiated recipient mice, which was reversible by pretransplant administration of antagonists of the CXCL12 receptor, CXCR4. Such agents are currently clinically available, suggesting that this approach could be used to improve stem cell transplantation and engraftment.

Adult↗
PubMed2005

A rapid method for retrovirus-mediated identification of complementation groups in Fanconi anemia patients.

Fanconi anemia (FA) is a rare autosomal recessive disorder that results from mutations in at least 11 different genes. Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intragenic variations, emphasizing the importance of identifying the complementation groups. In the present study we have employed bicistronic retrovirus vectors that coexpress FA-specific cDNAs for complementation groups A, C, F, and G, together with the enhanced green fluorescence protein (EGFP), allowing for specific analysis of transduced EGFP+ cells within bulk cultures by flow cytometry. In addition, the assay relies on the correction of the characteristic FA-associated G2/M arrest after treatment of cells with DNA-damaging agents, which is analyzed by flow cytometry. Results obtained with this assay matched the complementation groups known for 12 control lymphoblast cell lines tested. We report here the results obtained for 48 FA patients with unknown complementation groups using this new assay. Complementation groups were identified for 24 patients. We have identified mutations in the genes corresponding to the assigned complementation group in 23 samples. This assay has now been established in a standardized fashion for complementation assignments in FA patients and the subsequent directing of rapid mutation analysis in those patients.

Cell Line↗
PubMed2005

Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization.

The molecular events that regulate engraftment and mobilization of hematopoietic stem cells and progenitors (HSC/Ps) are still incompletely defined. We have examined the role of the Rho GTPases Rac1 and Rac2 in HSC engraftment and mobilization. Rac1, but not the hematopoietic-specific Rac2, is required for the engraftment phase of hematopoietic reconstitution, because Rac1(-/-) HSCs did not rescue in vivo hematopoiesis after transplantation, but deletion of Rac1 after engraftment did not impair steady-state hematopoiesis. Rac1(-/-) HSC/Ps showed impaired spatial localization to the endosteum but near-normal homing to the medullary cavity in vivo. Interaction with the bone marrow microenvironment in vitro was markedly altered. Whereas post-engraftment deletion of Rac1 alone did not impair hematopoiesis, deficiency of both Rac1 and Rac2 led to massive mobilization of HSCs from the marrow associated with ineffective hematopoiesis and intense selection for Rac-expressing HSCs. This mobilization was reversible by re-expression of Rac1. In addition, a rationally designed, reversible small-molecule inhibitor of Rac activation led to transient mobilization of engraftable HSC/Ps. Rac proteins thus differentially regulate engraftment and mobilization phenotypes, suggesting that these biological processes and steady-state hematopoiesis are biochemically separable and that Rac proteins may be important molecular targets for stem cell modification.

Aminoquinolines↗
PubMed2003

Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases.

The Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 are critical signaling regulators in mammalian cells. The deletion of both Rac1 and Rac2 murine alleles leads to a massive egress of hematopoietic stem/progenitor cells (HSC/Ps) into the blood from the marrow, whereas Rac1-/- but not Rac2-/- HSC/Ps fail to engraft in the bone marrow of irradiated recipient mice. In contrast, Rac2, but not Rac1, regulates superoxide production and directed migration in neutrophils, and in each cell type, the two GTPases play distinct roles in actin organization, cell survival, and proliferation. Thus, Rac1 and Rac2 regulate unique aspects of hematopoietic development and function.

Actins↗
PubMedDate not supplied

Bone marrow connexin-43 expression is critical for hematopoietic regeneration after chemotherapy.

Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junction channels (connexons) are formed by polypeptides (connexins) arranged in hexamers and represent the best described intercellular communication system. Connexin-43 (Cx43) is expressed by BM OS cells and has been associated with the cadherin/beta -catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Here, we employed an inducible gene-targeted murine approach to study the role of Cx43 in HSC proliferation and differentiation in vivo. Mx-Cre/Cx43+/+ and Mx-Cre/Cx43flox/flox littermates have been analyzed after gene deletion induced in vivo by the interferon-inducer poly (I)-poly (C), generating control (Cx43+) and Cx43-deficient (Cx43-/-) mice. After one week, Cx43+ and Cx43-/- mice were treated with 5-fluorouracil (5-FU). Cx43 expression in Cx43-/- BM was markedly reduced (> 90%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5- to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units (CFU-GM-), erythroid burst forming units (BFU-E) and mixed colony forming units (CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin-/c-kit+/Sca1+ cells in Cx43-/- BM was maintained when compared to Cx43+ BM. Short-term competitive repopulation ability of Cx43-/- BM cells was diminished as compared to Cx43+ mice, specifically for myeloid and B lymphoid cells, but showed spared long-term competitive repopulation ability with roughly normal hematopoietic differentiation. These data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.

Animals↗