Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In patients with chronic myeloid leukemia (CML), the leukemic (BCR-ABL+/Ph+) clone typically includes cells belonging to all of the myeloid lineages and frequently some B cells. From such observations it has been inferred that the initial BCR-ABL gene rearrangement event occurs in a pluripotent hematopoietic stem cell and that the clone subsequently generated is maintained by a subpopulation of neoplastic, BCR-ABL-expressing cells that retain at least some of the defining properties of normal hematopoietic stem cells. To test this hypothesis directly, we isolated various subpopulations of CD34+ cells from fresh or cryopreserved samples of peripheral blood from 5 CML patients with high white blood cell counts, 4 of which were selected because of their exclusive content of Ph+ progenitors (both colony-forming cells and long-term culture-initiating cells [LTC-IC]). Cells in each of the CD34+ subpopulations isolated were examined for the presence of BCR-ABL mRNA using a reverse transcriptase-polymerase chain reaction technique that reproducibly gave a positive signal from single K562 cells. BCR-ABL mRNA was detected in 117 of 147 samples (80%) in which actin mRNA was demonstrable. This included 60% to 90% of a large number of individually analyzed CD34+ cells including 46 single CD34+CD71-CD38- cells and 27 single CD34+CD71+CD38+ cells from 3 patients. In 2 of these cases, the same populations also contained a very high frequency of Ph+ LTC-IC. Our findings demonstrate BCR-ABL gene expression in neoplastic cells with functional as well as surface marker characteristics of very primitive normal hematopoietic cells. This implicates the BCR-ABL gene product directly in the acquisition by these cells of properties that alter their interactions with the microenvironment and deregulate their proliferation control.
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PMID:BCR-ABL expression in different subpopulations of functionally characterized Ph+ CD34+ cells from patients with chronic myeloid leukemia. 878 37

Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.
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PMID:Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. 922 64

CD34+ and CD34+ DR- cells from the bone marrow (BM) of chronic-phase chronic myelogenous leukaemia (CML) patients at diagnosis were tested for their colony-forming ability in response to early and intermediate-late colony stimulating factors (CSFs). Molecular analysis revealed that 55.6+/-9% SD of CD 34+ DR- colonies, in which actin and ABL mRNA were detectable, expressed the product of the BCR-ABL gene. The percentage and the clonogenic efficiency of CML DR- cells were significantly lower than those of comparable DR- cells from normal donors. However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, IL-11 and IL-3, used as single factors in the presence of erythropoietin (EPO) and was almost entirely due to erythroid progenitors. Conversely, optimal stimulation of CD34 +DR- cells from normal donors required co-incubation with three or more CSFs. Stroma-noncontact long-term cultures were then established in the presence of exogenous CSFs and human irradiated allogeneic stromal layers or the murine stromal cell line M2-10B4, engineered to produce G-CSF and IL-3. In these cultures the combination of SCF and IL-3 induced a 25.4 +/- 5 SD, 40 +/- 6 SD and 20.5 +/- 6 SD fold increase of colony-forming unit cells (CFU-C), at weeks 2, 4 and 5, respectively. At the same time-points the number of primitive long-term culture initiating cells (LTC-IC) showed a 4 +/- 2 SD, 3.3 +/- 1.5 SD and 2.3 +/-1 SD fold increase compared to baseline values. BCR-ABL mRNA analysis of single colonies demonstrated that 27 +/- 9% SD and 7 +/- 3% SD CFU-C at weeks 4 and 5, respectively, expressed the fusion gene, whereas leukaemic LTC-IC disappeared from the culture by week 2. These results suggest that leukaemic CD34+ DR- cells have a different pattern of response to CSFs than normal cells. In addition, we established culture conditions which allow selective expansion of benign haemopoietic cells coexisting with leukaemic progenitors.
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PMID:Selective expansion of normal haemopoietic progenitors from chronic myelogenous leukaemia marrow. 957 92

We have tested whether peripheral blood mononuclear cells (PBMNCs) from interferon (IFN)-treated patients may lose residual BCR-ABL sequence-positive progenitor cells when long-term cultured for 35 days on allogeneic stromal cells. IFN-treated patients have low white blood cell counts and a fair number of BCR-ABL-negative colony-forming cells in the peripheral blood. Particularly, IFN responders show increased numbers of normal hematopoietic cells. We have quantitatively analyzed progenitor cells in PBMNCs of IFN-treated patients by combining the clonogenic assay in semisolid medium with interphase fluorescent in situ hybridization (FISH). Thus, the identification is possible of the BCR-ABL status of colony-forming progenitor cells. In IFN-treated patients, the number of BCR-ABL-positive CFCs is considerably decreased and BCR-ABL-negative CFCs appear in the peripheral blood. We could show that after LTC for 35 days of the same PBMNCs on irradiated allogeneic normal stromal cells residual BCR-ABL sequence-positive CFCs were still present. In some cases the relative number of BCR-ABL sequence-positive CFCs was found to be increased after LTC. A minor proportion of blood samples from IFN-treated patients did not give rise to CFCs after LTC on allogeneic stromal cells (three of 10 patients). Inter- and intraindividual variations can be found with regard to loss or gain of BCR-ABL sequence-positive colonies after LTC. We conclude that early CML progenitor cells persist in the peripheral blood of IFN-treated patients and that a certain proportion may survive long-term culture.
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PMID:Does long-term culture favor normal clonogenic cells from interferon-treated patients with chronic myelogenous leukemia? 1023 67

The growth factor combination containing early acting cytokines FLT-3 ligand (FL), Stem Cell Factor (SCF) and thrombopoietin (TPO) is able to maintain, for an extended culture period, early stem cells, defined as long-term repopulating NOD/SCID mice (Scid Repopulating Cell-SRC) contained in cord blood (CB). In this culture system, the role of IL-6 and IL-3 has not been clearly established. Using a combination of FL+TPO+SCF with or without IL-6, we were able to form CB CD34+ cells for 30 weeks. The CB CD34+ cells cultured in this system engrafted NOD/SCID mice after 6 weeks of culture; the cells from primary recipients were also able to engraft secondary NOD/SCID mice. When CB CD34+ cells were cultured in the presence of IL-3 in the place of IL-6 we observed an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture. However, more primitive LTC-IC output increased up to week 6 with the growth factor combination containing IL-3 and then decreased and disappeared, while with the growth factor combination with or without IL-6 increased up to week 23. Cells cultured for 4 weeks with the 4-factor combination containing IL-3 engrafted NOD/SCID mice less efficiently. Repopulation of NOD/SCID mice was no longer observed when ex vivo expansion was performed for 6 weeks. This study provides some evidence that no differences could be detected in long-term maintenance and even expansion of human primitive cord blood cells cultured with FL+TPO+SCF in the presence or absence of IL-6. Under the culture conditions employed in this study, the presence of IL-3 reduced the repopulating potential of expanded CB CD34+ cells.
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PMID:Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model. 1138 43

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.
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PMID:NOD/SCID repopulating cells but not LTC-IC are enriched in human CD34+ cells expressing the CCR1 chemokine receptor. 1145 79

The objective of this study was to analyze the mobilization kinetics of normal (BCR-ABL(neg)) and malignant (BCR-ABL(pos)) progenitor cells using a new, low toxic, out-patient-based mobilization regimen for Philadelphia chromosome-positive (Ph(pos)) chronic myelogenous leukemia (CML) patients. High doses of hydroxyurea (HD-HU, 3.5 g/m(2) per day, orally for 7 days) followed by granulocyte colony-stimulating factor (G-CSF) (10 microg/kg subcutaneously) were administered to 11 newly diagnosed CML patients. Each apheresis product (n = 30) was individually analyzed for the number and genotype of mature colony-forming cells (CFC) and primitive long-term culture initiating cells (LTC-IC), respectively, by reverse transcription polymerase chain reaction (RT-PCR) of individual colonies. Sufficient numbers of CD34(+) cells/kg bodyweight (BW) could easily be obtained in all patients (median, 15 x 10(6)/kg BW per patient) with a median number of three aphereses performed per patient (range 2-4). Almost each apheresis itself (25/30) contained > or =2 x 10(6) CD34(+) cells/kg BW. All patients with low and intermediate Sokal risk indices (9/11) mobilized primarily BCR-ABL(neg) LTC-IC (median 92%, range 47-100) and CFC (median 89%, range 57-100). Moreover, the mean percentage of BCR-ABL(neg) CFC and LTC-IC in the various apheresis products in these patients did not change throughout the entire time of hematopoietic regeneration. The toxicity of the mobilization procedure was low. Side effects were mild erythema in 8/11 and oral mucositis in 3/11 patients. Overall, the low toxicity of this regimen, together with the fact that sufficient BCR-ABL(neg) progenitors can be collected throughout the entire period of hematopoietic regeneration, renders this mobilization regimen particularly attractive for the collection of BCR-ABL(neg) progenitors in early chronic phase of Ph(pos) CML.
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PMID:High-dose hydroxyurea plus G-CSF mobilize BCR-ABL-negative progenitor cells (CFC, LTC-IC) into the blood of newly diagnosed CML patients at any time of hematopoietic regeneration. 1198

To study the hierarchical levels of stem cell targets for ABL-kinase domain mutations in CML, highly purified CD34+CD38- and CD34+CD38+ cell populations and their LTC-IC-derived progeny were analyzed in four patients at diagnosis (n=1) or in advanced phases (n=3) of their disease. In the single patient with early phase CML who later developed an Imatinib Mesylate-resistance and a Y253H mutation, no mutation was detectable in purified cell fractions analyzed at diagnosis nor in their LTC-IC-derived progeny. In contrast, in three patients in advanced phase CML, ABL-kinase mutations demonstrated in peripheral blood cells by sequencing (Q252E and M351T) were detectable in the FACS-sorted cells and became amplified in the LTC-IC-derived progeny of the primitive cells. These findings demonstrate that in late CP or advanced CML, ABL-kinase mutations occur as an intraclonal event in the primitive Ph1+ stem cell compartments with progression of this clone towards IM-resistant blast phase.
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PMID:Evidence of ABL-kinase domain mutations in highly purified primitive stem cell populations of patients with chronic myelogenous leukemia. 1538 Oct 60

A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34+ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34+ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.
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PMID:Support of hMSCs transduced with TPO/FL genes to expansion of umbilical cord CD34+ cells in indirect co-culture. 1668 32

Cytokines and chemokines control hematopoietic stem and progenitor cell (HPC) proliferation and trafficking. However, the role of nonpeptide mediators in the bone marrow microenvironment has remained elusive. Particularly CysLT(1), a G protein-coupled receptor recognizing inflammatory mediators of the cysteinyl leukotriene family, is highly expressed in HPCs. We therefore analyzed the effects of its ligands on human CD34(+) HPCs. The most potent CysLT(1) ligand, LTD(4), rapidly and significantly up-regulated alpha(4)beta(1) and alpha(5)beta(1) integrin-dependent adhesion of both primitive and committed HPC. LTD(4)-triggered adhesion was inhibited by specific CysLT(1) antagonists. The effects of other CysLT(1) ligands were weak (LTC(4)) or absent (LTE(4)). In serum-free liquid cultures supplemented with various hematopoietic cytokines including IL-3, only LTD(4) significantly augmented the expansion of HPCs in a dose-dependent manner comparable to that of peptide growth factors. LTC(4) and LTE(4) were less effective. In CD34(+) cell lines and primary HPCs, LTD(4) induced phosphorylation of p44/42 ERK/MAPK and focal adhesion kinase-related tyrosine kinase Pyk2, which is linked to integrin activation. Bone marrow stromal cells produced biologically significant amounts of cysteinyl leukotrienes only when hematopoietic cells were absent, suggesting a regulatory feedback mechanism in the hematopoietic microenvironment. In contrast to antagonists of the homing-related G protein-coupled receptor CXCR4, administration of a CysLT(1) antagonist failed to induce human CD34(+) HPC mobilization in vivo. Our results suggest that cysteinyl leukotriene may contribute to HPC retention and proliferation only when cysteinyl leukotriene levels are increased either systemically during inflammation or locally during marrow aplasia.
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PMID:The CysLT1 ligand leukotriene D4 supports alpha4beta1- and alpha5beta1-mediated adhesion and proliferation of CD34+ hematopoietic progenitor cells. 1945 74


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