Biomedical subjects

G Q Daley

Publications and source records attributed to G Q Daley.

At least 19 recordsLinked to original sources

PubMed2006

Stem cells and their niche: a matter of fate.

Embryonic stem cells provide an in vitro model for developmental biologists to study cell fate decisions during ontogenesis, while somatic stem cells allow physiologists to understand tissue homeostasis in the adult. The behavior of stem cells is dependent on an intimate relationship with a supportive niche. This brief review highlights some of the most important recent trends in stem cell biology, focusing in particular on the supportive microenvironments for both embryonic and adult stem cells. Known intrinsic and extrinsic molecular players from the best-characterized stem cell types are summarized, illuminating a number of shared environmental cues among tissues originating from all three embryonic germ layers.

Animals↗

PubMed2006

Progress and prospects: gene transfer into embryonic stem cells.

With the isolation of human embryonic stem cells (hESCs) in 1998 came the realization of a long-sought aspiration for an unlimited source of human tissue. The difficulty of differentiating ESCs to pure, clinically exploitable cell populations to treat genetic and degenerative diseases is being solved in part with the help of genetically modified cell lines. With progress in genome editing and somatic cell nuclear transfer, it is theoretically possible to obtain genetically repaired isogenic cells. Moreover, the prospect of being able to select, isolate and expand a single cell to a vast population of cells could achieve a unique level of quality control, until now unattainable in the field of gene therapy. Most of the tools necessary to develop these strategies already exist in the mouse ESC system. We review here the advances accomplished in those fields and present some possible applications to hESC research.

Animals↗

PubMed2003

Treatment by design in leukemia, a meeting report, Philadelphia, Pennsylvania, December 2002.

Novel approaches have been designed to treat leukemia based on our understanding of the genetic and biochemical lesions present in different malignancies. This meeting report summarizes some of the recent advances in leukemia treatment. Based on the discoveries of cellular oncogenes, chromosomal translocations, monoclonal antibodies, multidrug resistance pumps, signal transduction pathways, genomics/proteonomic approaches to clinical diagnosis and mutations in biochemical pathways, clinicians and basic scientists have been able to identify the particular genetic mutations and signal transduction pathways involved as well as design more appropriate treatments for the leukemia patient. This meeting report discusses these exciting new therapies and the results obtained from ongoing clinical trials. Furthermore, rational approaches to treat complications of tumor lysis syndrome by administration of the recombinant urate oxidase protein, also known as rasburicase, which corrects the biochemical defect present in humans, were discussed. Clearly, over the past 25 years, molecular biology and biotechnology has provided the hematologist/oncologist novel bullets in their arsenal that will allow treatment by design in leukemia.

Antineoplastic Agents↗

PubMed2001

Single nucleotide polymorphisms in multiple novel thrombospondin genes may be associated with familial premature myocardial infarction.

BACKGROUND: Recent advances in high-throughput genomics technology have expanded our ability to catalogue allelic variants in large sets of candidate genes related to premature coronary artery disease. METHODS AND RESULTS: A total of 398 families were identified in 15 participating medical centers; they fulfilled the criteria of myocardial infarction, revascularization, or a significant coronary artery lesion diagnosed before 45 years in men or 50 years in women. A total of 62 vascular biology genes and 72 single-nucleotide polymorphisms were assessed. Previously undescribed variants in 3 related members of the thrombospondin protein family were prominent among a small set of single-nucleotide polymorphisms that showed a statistical association with premature coronary artery disease. A missense variant of thrombospondin 4 (A387P) showed the strongest association, with an adjusted odds ratio for myocardial infarction of 1.89 (P=0.002 adjusted for covariates) for individuals carrying the P allele. A variant in the 3' untranslated region of thrombospondin-2 (change of thymidine to guanine) seemed to have a protective effect against myocardial in individuals homozygous for the variant (adjusted odds ratio of 0.31; P=0.0018). A missense variant in thrombospondin-1 (N700S) was associated with an adjusted odds ratio for coronary artery disease of 11.90 (P=0.041) in homozygous individuals, who also had the lowest level of thrombospondin-1 by plasma assay (P=0.0019). CONCLUSIONS: This large-scale genetic study has identified the potential of multiple novel variants in the thrombospondin gene family to be associated with familial premature myocardial infarction. Notwithstanding multiple caveats, thrombospondins specifically and high-throughput genomic technology in general deserve further study in familial ischemic heart disease.

Adult↗

PubMed2001

Cooperative and redundant effects of STAT5 and Ras signaling in BCR/ABL transformed hematopoietic cells.

The Akt, Ras and STAT5 signaling pathways have each been linked to transformation of hematopoietic cells by BCR/ABL. However the relative contributions of these signaling pathways to BCR/ABL mediated cytokine-independent survival, proliferation and resistance to DNA damage-induced apoptosis have not been systematically defined. Here we report that activation of either Akt, Ras or STAT5 confers cytokine-independent survival to IL-3 dependent BaF3 cells. Ras or STAT5, but not Akt, also drives cytokine-independent proliferation and imparts sustained resistance to DNA damage-induced apoptosis. We also show that dominant negative (DN) inhibition of STAT5, but not Ras or Akt, significantly reduces resistance to DNA damage-induced apoptosis in BCR/ABL transformed BaF3 cells. Whereas inhibition of STAT5 or Ras alone does not compromise cytokine-independent proliferation of BaF3-BCR/ABL cells, simultaneous blockade of both STAT5 and Ras reduces proliferation and maximally sensitizes BaF3-BCR/ABL cells to DNA damage induced by gamma-irradiation, suggesting a cooperative role for these two signaling pathways in BCR/ABL transformation. The anti-apoptotic properties of BCR/ABL can be partly explained by an increase in the expression of Pim-1 and Bcl-XL, as ectopic expression of these STAT5 target genes imparts both cytokine-independent survival and partial gamma-radiation resistance. These data illustrate both cooperative and redundant effects of STAT5 and Ras signaling in BCR/ABL transformed cells, with STAT5 playing a dominant role in resistance to DNA damage-induced apoptosis.

Animals↗

PubMed2001

Expression of interferon consensus sequence binding protein induces potent immunity against BCR/ABL-induced leukemia.

Mice deficient in the interferon consensus sequence binding protein (ICSBP) develop a disease resembling chronic myeloid leukemia (CML), which in humans is caused by the BCR/ABL oncoprotein. Interferon-alpha (IFN-alpha) induces ICSBP expression and is an effective therapy for CML. This study examined whether enforced expression of ICSBP might antagonize BCR/ABL-induced leukemia; results demonstrated that ICSBP-modified cells generated a protective CD8(+) cytotoxic T-cell response against BCR/ABL-transformed BaF3 cells in a murine leukemia model. ICSBP expression represents a novel means of stimulating a host immune response to BCR/ABL(+) leukemia cells and a potential strategy for immunotherapy of CML. (Blood. 2001;97:3491-3497)

Animals↗

PubMed2001

Autocrine and paracrine effects of an ES-cell derived, BCR/ABL-transformed hematopoietic cell line that induces leukemia in mice.

During differentiation in vitro, Embryonic Stem (ES) cells generate both primitive erythroid and definitive myeloid lineages in a process that mimics hematopoiesis in the mammalian yolk sac. To investigate leukemic transformation of these embryonic hematopoietic progenitors, we infected differentiating cultures of ES cells with the Chronic Myeloid Leukemia-specific BCR/ABL oncoprotein. Following a period of liquid culture, we isolated two transformed subclones, EB57 and EB67, that retained characteristics of embryonic hematopoietic progenitors and induced a fatal leukemia in mice characterized by massive splenomegaly and granulocytosis. Histopathology of the spleen revealed an abundance of undifferentiated blast-like cells. Investigation of the clonal origins of the granulocytes in the peripheral blood demonstrated that the injected donor cells contributed modestly to the granulocyte population while the majority were host-derived. EB57 secretes IL-3 and unidentified cytokines that can stimulate autocrine and paracrine cell proliferation, presumably accounting for the reactive granulocytosis in diseased mice. These BCR/ABL transformed hematopoietic derivatives of ES cells recapitulate the relationship of BCR/ABL expression to IL-3 production that has been described for primitive hematopoietic progenitors from human CML patients, and illustrates the potential for autocrine and paracrine effects of BCR/ABL-infected cells in murine models.

Animals↗

PubMed2001

Treatment of Bcr/Abl-positive acute lymphoblastic leukemia in P190 transgenic mice with the farnesyl transferase inhibitor SCH66336.

The Philadelphia (Ph) chromosome is found in approximately 3% of pediatric patients with acute lymphoblastic leukemia (ALL) and the percentage markedly increases in adult patients. The prognosis for this class of patients is poor, and no standard chemotherapy combination so far has demonstrated long-term efficacy. The Ph-translocation joins the BCR and ABL genes and leads to expression of a chimeric Bcr/Abl protein with enhanced tyrosine kinase activity. This increase in activity leads to malignant transformation by interference with basic cellular functions such as the control of proliferation, adherence to stroma and extracellular matrix, and apoptosis. One important pathway activated by Bcr/Abl is the Ras pathway. Ras proteins have to undergo a series of posttranslational modifications to become biologically active. The first modification is the farnesylation of the C-terminus catalyzed by farnesyl transferase. We studied the effect of the farnesyl transferase inhibitor SCH66336 in an in vivo murine model of Bcr/Abl-positive acute lymphoblastic leukemia. In the early leukemic phase, mice were randomly assigned to a treatment, a vehicle, and a nontreatment group. The treatment was well tolerated without any detectable side effects. All animals of the control groups died of leukemia/lymphoma within 103 days (range, 18-103 days). In contrast, 80% of the drug-receiving group survived without any signs of leukemia or lymphoma until termination of treatment, after a median treatment period of 200 days (range, 179-232 days). We conclude that farnesyl transferase inhibitor SCH66336 is able to revert early signs of leukemia and significantly prolongs survival in a murine ALL model.

Alkyl and Aryl Transferases↗

PubMed2001

Activity of the farnesyl protein transferase inhibitor SCH66336 against BCR/ABL-induced murine leukemia and primary cells from patients with chronic myeloid leukemia.

BCR/ABL, the oncoprotein responsible for chronic myeloid leukemia (CML), transforms hematopoietic cells through both Ras-dependent and -independent mechanisms. Farnesyl protein transferase inhibitors (FTIs) were designed to block mutant Ras signaling, but they also inhibit the growth of transformed cells with wild-type Ras, implying that other farnesylated targets contribute to FTI action. In the current study, the clinical candidate FTI SCH66336 was characterized for its ability to inhibit BCR/ABL transformation. When tested against BCR/ABL-BaF3 cells, a murine cell line that is leukemogenic in mice, SCH66336 potently inhibited soft agar colony formation, slowed proliferation, and sensitized cells to apoptotic stimuli. Quantification of activated guanosine triphosphate (GTP)-bound Ras protein and electrophoretic mobility shift assays for AP-1 DNA binding showed that Ras effector pathways are inhibited by SCH66336. However, SCH66336 was more inhibitory than dominant-negative Ras in assays of soft agar colony formation and cell proliferation, suggesting activity against targets other than Ras. Cell cycle analysis of BCR/ABL-BaF3 cells treated with SCH66336 revealed G2/M blockade, consistent with recent reports that centromeric proteins that regulate the G2/M checkpoint are critical farnesylated targets of FTI action. Mice injected intravenously with BCR/ABL-BaF3 cells developed acute leukemia and died within 4 weeks with massive splenomegaly, elevated white blood cell counts, and anemia. In contrast, nearly all mice treated with SCH66336 survived and have remained disease-free for more than a year. Furthermore, SCH66336 selectively inhibited the hematopoietic colony formation of primary human CML cells. As an oral, nontoxic compound with a mechanism of action distinct from that of ABL tyrosine kinase inhibition, FTI SCH66336 shows promise for the treatment of BCR/ABL-induced leukemia.

Alkyl and Aryl Transferases↗

PubMed2001

The heart SNPs a beat: polymorphisms in candidate genes for cardiovascular disease.

Several environmental risk factors of cardiovascular disease are well established, but genetic risk alleles contributing to the disease in the general population are hotly debated. New strategies focusing on polymorphism discovery in candidate disease genes followed by tests of association to genes across the genome offer a pioneering approach to identifying risk alleles. Several hundred candidate genes for cardiovascular disease have been screened for common polymorphisms and these variants may provide susceptibility alleles which largely contribute to risk of cardiovascular disease in the general population. However, the impact of common susceptibility alleles for disease management will depend on many years of future investigation.

Cardiovascular Diseases↗

PubMed2001

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.

Animals↗

PubMed2000

Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation.

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.

Alkaline Phosphatase↗

PubMed2000

A genetic screen to identify genes that rescue the slow growth phenotype of c-myc null fibroblasts.

The c-myc gene is frequently over-expressed in human cancers and is involved in regulation of proliferation, differentiation and apoptosis. c-Myc is a transcription factor that acts primarily by regulating the expression of other genes. However, it has been very difficult to identify bona fide c-Myc target genes that explain its diverse biological activities. The recent generation of c-myc deficient Rat1A fibroblasts with a profound and stable growth defect provides a new system to search for genes that can substitute for c-myc in proliferation. In this study, we have attempted to identify genes that rescue the slow growth phenotype of c-myc null cells through introduction of a series of potent cell cycle regulatory genes and several retroviral cDNA expression libraries. None of the candidate genes tested, including SV40 T-antigen and adenovirus E1A, caused reversal of the c-myc null growth defect. Furthermore, extensive screens with high-complexity retroviral cDNA libraries from three different tissue sources revealed that only c-myc and N-myc rescued the c-myc null slow-growth phenotype. Our data support the notion that there are no functional equivalents of the myc family of proto-oncogenes and also suggest that there are no c-Myc-activated genes that alone can substitute for c-Myc in control of cell proliferation.

Adenovirus E1A Proteins↗

PubMed2000

Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers.

To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.

Adenocarcinoma↗

PubMed2000

Mining for SNPs: putting the common variants--common disease hypothesis to the test.

Classical molecular genetic strategies have succeeded in identifying mutations responsible for numerous rare diseases with Mendelian patterns of inheritance, but have been largely unsuccessful in unravelling the (genetic basis of complex medical conditions like cardiovascular disease' diabetes and mental illness. These common disorders are shaped by multiple genes that exert weak allelic effects in the setting of confounding environmental variables. Association study designs provide statistical povwer to reveal the modest contributions of weak alleles, and evidence is mounting that common genetic polymorphisms play a role in complex diseases. Cataloguing genetic variation in human populations is a prerequisite for further validation of the 'common variants-common disease' hypothesis, and polymorphism discovery has begun in earnest in the academic and private sector. We will review several strategies for high-throughput polymorphism discovery and discuss the implications of early results from polymorphism screens for future genetic studies.

Gene Frequency↗

PubMed1999

The BCR/ABL oncogene alters the chemotactic response to stromal-derived factor-1alpha.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is a chemoattractant for CD34(+) progenitor cells, in vitro and in vivo. The receptor for SDF-1alpha, CXCR-4, is a 7 transmembrane domain receptor, which is also a coreceptor for human immunodeficiency virus (HIV). Here we show that transformation of hematopoietic cell lines by BCR/ABL significantly impairs their response to SDF-1alpha. Three different hematopoietic cell lines, Ba/F3, 32Dcl3, and Mo7e, were found to express CXCR-4 and to respond to SDF-1alpha with increased migration in a transwell assay. In contrast, after transformation by the BCR/ABL oncogene, the chemotactic response to SDF-1alpha was reduced in all 3 lines. This effect was directly due to BCR/ABL, because Ba/F3 cells, in which the expression of BCR/ABL could be regulated by a tetracycline-inducible promoter, also had reduced chemotaxis to SDF-1alpha when BCR/ABL was induced. The reduced response to SDF-1alpha was not due to an inability of BCR/ABL-transformed cell lines to migrate in general, as spontaneous motility of BCR/ABL-transformed cells was increased. In mice, injection of SDF-1alpha into the spleen resulted in a transient accumulation of untransformed Ba/F3 cells, but not Ba/F3. p210(BCR/ABL) cells administered simultaneously. The mechanism may involve inhibition of CXCR-4 receptor function, because in BCR/ABL-transformed cells, CXCR-4 receptors were expressed on the cell surface, but SDF-1alpha calcium flux was inhibited. Because SDF-1alpha and CXCR-4 are felt to be involved in progenitor cell homing to marrow, the abnormality decribed here could contribute to the homing and retention defects typical of immature myeloid cells in chronic myelogenous leukemia.

Animals↗

PubMed1999

BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells.

Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo(-/-) and EpoR(-/-) mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210(BCR-ABL) and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR(-/-) mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.

Animals↗

PubMed1999

The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity.

The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3-dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)-like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)-treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non- 5-FU-treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.

Animals↗