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Gerard Brady

Publications and source records attributed to Gerard Brady.

7 recordsLinked to original sources
PubMed2006

Notch signaling induces apoptosis in primary human CD34+ hematopoietic progenitor cells.

Notch signaling regulates diverse cell fate decisions during development and is reported to promote murine hematopoietic stem cell (HSC) self-renewal. The purpose of this study was to define the functional consequences of activating the Notch signaling pathway on self-renewal in human HSCs. Subsets of human umbilical cord blood CD34(+) cells were retrovirally transduced with the constitutively active human Notch 1 intracellular domain (N1ICD). N1ICD-transduced cells proliferated to a lesser extent in vitro than cells transduced with vector alone, and this was accompanied by a reduction in the percentage and absolute number of CD34(+) cell populations, including CD34(+)Thy(+)Lin(-) HSCs. Ectopic N1ICD expression inhibited cell cycle kinetics concurrent with an upregulation of p21 mRNA expression and induced apoptosis. Transduction of cells with HES-1, a known transcriptional target of Notch signaling and a mediator of Notch function, had no effect on HSC proliferation, indicating that the mechanism of the Notch-induced effect is HES-1-independent. The results of this study show that activation of the Notch signaling pathway has an inhibitory effect on the proliferation and survival of human hematopoietic CD34(+) cells populations. These findings have important implications for strategies aimed at promoting self-renewal of human HSCs.

Antigens, CD34↗
PubMed2005

Routine expression profiling of microarray gene signatures in acute leukaemia by real-time PCR of human bone marrow.

Cancer subtype diagnosis using microarray signatures has the potential to transform pathological diagnosis but the routine measurement of genes signatures remains difficult. Reverse transcription polymerase chain reaction (RT-PCR) measurement of Indicator genes for acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) was used to determine gene signatures. Bone marrow (BM) mononuclear cells were sorted into total, CD34(+) and CD34(-) fractions, and mRNAs globally amplified from each fraction using polyA PCR. The expression profile of the 17 top-ranked genes distinguishing AML and ALL were measured by RT-PCR in five ALL, 26 AML, 12 AML remission, four chronic myeloid leukaemia (CML) and nine morphologically normal BM samples. All but two of the genes measured showed similar expression in AML and ALL to that reported previously. Specifically, c-MYB (P </= 0.04) was significantly increased in ALL in the total fraction, whilst HOXA9 (P </= 0.19) and cystatin c (P </= 0.01) were increased in AML in the CD34(+) and CD34(-) fractions, respectively. c-MYB, hSNF2, RBAP48, HKRT-1, LYN, CD33, Adipsin and HOXA9 were increased in AML compared with remission AML, indicating an ability to determine disease activity. The method used is simple, sensitive and robust, enabling routine clinical use, and it can also be extended to other tumours types with gene signatures.

Acute Disease↗
PubMed2003

Thyroid hormone activates fibroblast growth factor receptor-1 in bone.

Thyroid hormone (T3) and the T3 receptor (TR) alpha gene are essential for bone development whereas adult hyperthyroidism increases the risk of osteoporotic fracture. We isolated fibroblast growth factor receptor-1 (FGFR1) as a T3-target gene in osteoblasts by subtraction hybridization. FGFR1 mRNA was induced 2- to 3-fold in osteoblasts treated with T3 for 6-48 h, and FGFR1 protein was stimulated 2- to 4-fold. Induction of FGFR1 was independent of mRNA half-life and abolished by actinomycin D and cycloheximide, indicating the involvement of an intermediary protein. Fibroblast growth factor 2 (FGF2) stimulated MAPK in osteoblasts, and pretreatment with T3 for 6 h induced a more rapid response to FGF that was increased in magnitude by 2- to 3-fold. Similarly, T3 enhanced FGF2-activated autophosphorylation of FGFR1, but did not modify FGF2-induced phosphorylation of the docking protein FRS2. These effects were abolished by the FGFR-selective inhibitors PD166866 and PD161570. In situ hybridization analyses of TRalpha-knockout mice, which have impaired ossification and skeletal mineralization, revealed reduced FGFR1 mRNA expression in osteoblasts and osteocytes, whereas T3 failed to stimulate FGFR1 mRNA or enhance FGF2-activated MAPK signaling in TRalpha-null osteoblasts. These findings implicate FGFR1 signaling in T3-dependent bone development and the pathogenesis of skeletal disorders resulting from thyroid disease.

Animals↗
PubMed2003

Identification of a putative intestinal stem cell and early lineage marker; musashi-1.

There are few reliable markers for adult stem cells and none for those of the intestinal epithelium. Previously, indirect experimental approaches have predicted stem cell position and numbers. The Musashi-1 (Msi-1) gene encodes an RNA binding protein associated with asymmetric divisions in neural progenitor cells. Two-day-old, adult, and 4.5 h, 1-, 2-, 4- and 12-day post-irradiation samples of BDF1 mouse small intestine, together with some samples of mouse colon were stained with a rat monoclonal antibody to Musashi-1 (14 H-1). Min ( + / - ) mice with small intestinal adenomas of varying sizes were also analysed. Samples of human small and large bowel were also studied but the antibody staining was weak. Musashi-1 expression was observed using immunohistochemistry in neonatal, adult, and regenerating crypts with a staining pattern consistent with the predicted number and distribution of early lineage cells including the functional stem cells in these situations. Early dysplastic crypts and adenomas were also strongly Musashi-1 positive. In situ hybridization studies showed similar expression patterns for the Musashi mRNA and real-time quantitative RT-PCR showed dramatically more Msi-1 mRNA expression in Min tumours compared with adjacent normal tissue. These observations suggest that Musashi-1 is a marker of stem and early lineage progenitor cells in murine intestinal tissue.

Adult↗
PubMed2002

Coexpression of parathyroid hormone related protein and its receptor in early breast cancer predicts poor patient survival.

PURPOSE: Parathyroid hormone-related protein (PTHRP) is in part responsible for the clinical syndrome of hypercalcaemia of malignancy and has been implicated as an important factor in the development of bone metastases. The aim of this study was to determine the coexpression of PTHRP and its receptor in early breast cancer (EBC) and bone metastases (BM), and correlate these findings to clinical outcome. EXPERIMENTAL DESIGN: Samples of surgically excised EBC (n = 176) and BM (n = 43) were collected and stored in liquid nitrogen. PTHRP protein was determined using immunohistochemistry and receptor mRNA using in situ hybridization (n = 107) or semiquantitative reverse transcription-PCR (n = 69). RESULTS: PTHRP protein was expressed in 115 of 170 (68%) EBC compared with 100% of BM (P < 0.001), whereas its receptor mRNA was expressed in 88 of 176 (50%) EBC compared with 35 of 43 (81%) BM (P < 0.001). Coexpression of both PTHRP and its receptor was present in 62 EBC samples (37%) and in 35 BM samples (81%; P < 0.001). The PTHRP receptor correlated well with increasing patient age, but not with tumor size, grade, estrogen receptor, progesterone receptor, or lymph node status. Individually PTHRP and PTHRP receptor both correlated well with a reduced disease-free survival (P < 0.004) and receptor alone with reduced overall survival (P < 0.003). Coexpression of both PTHRP and receptor predicted the worst clinical outcome at 5 years, with a mortality rate of 20 of 62 (32%) compared with the ligand and receptor-negative group with 2 of 32 (6%; P < 0.004). CONCLUSIONS: Overall these results show that the PTHRP receptor is expressed more frequently in BM than EBC, and is associated with poor clinical outcome and survival.

Aged↗
PubMed1995

Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells.

BACKGROUND: Many differentiating tissues contain progenitor cells that differ in their commitment states but cannot be readily distinguished or segregated. Molecular analysis is therefore restricted to mixed populations or cell lines which may also be heterogeneous, and the critical differences in gene expression that might determine divergent development are obscured. In this study, we combined global amplification of mRNA transcripts in single cells with identification of the developmental potential of processed cells on the basis of the fates of their sibling cells from clonal starts. RESULTS: We analyzed clones of from four to eight hemopoietic precursor cells which had a variety of differentiative potentials; sibling cells generally each formed clones of identical composition in secondary culture. Globally amplified cDNA was prepared from individual precursors whose developmental potential was identified by tracking sibling fates. Further cDNA samples were prepared from terminally maturing, homogeneous hemopoietic cell populations. Together, the samples represented 16 positions in the hemopoietic developmental hierarchy. Expression patterns in the sample set were determined for 29 genes known to be involved in hemopoietic cell growth, differentiation or function. The cDNAs from a bipotent erythroid/megakaryocyte precursor and a bipotent neutrophil/macrophage precursor were subtractively hybridized, yielding numerous differentially expressed cDNA clones. Hybridization of such clones to the entire precursor sample set identified transcripts with consistent patterns of differential expression in the precursor hierarchy. CONCLUSIONS: Tracking of sibling fates reliably identifies the differentiative potential of a single cell taken for PCR analysis, and demonstrates the existence of a variety of distinct and stable states of differentiative commitment. Global amplification of cDNA from single precursor cells, identified by sibling fates, yields a true representation of lineage- and stage-specific gene expression, as confirmed by hybridization to a broad panel of probes. The results provide the first expression mapping of these genes that distinguishes between progenitors in different commitment states, generate new insights and predictions relevant to mechanism, and introduce a powerful set of tools for unravelling the genetic basis of lineage divergence.

Animals↗