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W M Roberts

Publications and source records attributed to W M Roberts.

At least 37 records · Page 2Linked to original sources
PubMed1994

Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells.

Patients with solid tumors are increasingly being treated by autologous bone marrow transplantation (BMT). Although response rates appear to be increased, disease recurrence is the commonest cause of treatment failure. Whether relapse is entirely due to residual disease in the patient or arises also from infiltrating malignant cells contained in the autologous marrow transplant has not been resolved. If the latter explanation is correct, then purging would be required as part of the transplantation procedure. We used retrovirally mediated transfer of the neomycin-resistance gene to mark BM harvested from eight patients with neuroblastoma in clinical remission. The marked marrow cells were subsequently reinfused as part of an autologous BMT. At relapse, we sought the marker gene in malignant cell populations. Three patients have relapsed, and in each the marker gene was detected by phenotypic and genetic analyses of resurgent malignant cells at medullary and extramedullary sites. Analysis of neuroblast DNA for discrete marker gene integration sites suggested that at least 200 malignant cells, each capable of tumor formation, were introduced with the autologous marrow transplant and contributed to relapse. Thus, autologous BMTs administered to patients with this solid tumor may contain a multiplicity of malignant cells that subsequently contribute to relapse. The marker-gene technique we describe should permit evaluation of the mechanisms of relapse and the efficacy of purging in patients receiving autologous marrow transplantation for other solid tumors that infiltrate the marrow.

Base Sequence↗
PubMed1994

Intensive chemotherapy for Philadelphia-chromosome-positive acute lymphoblastic leukaemia.

Childhood acute lymphoblastic leukaemia with the classic Philadelphia chromosome translocation is fatal in patients treated with chemotherapy alone. We report probable cures in three adolescents and one child who received extensively reinforced, early chemotherapy followed by rotational treatment with pairs of non-cross-resistant drugs. The median duration of leukaemia-free survival in this subgroup is 6.5 years (range 6-8 years). The two patients with long-term bone marrow surveillance for residual disease showed no evidence of the Philadelphia chromosome at 31 and 53 months post-remission. Such intensive chemotherapy is a reasonable option for patients who are not able to undergo bone marrow transplantation.

Adolescent↗
PubMed1994

Calcium-triggered exocytosis and endocytosis in an isolated presynaptic cell: capacitance measurements in saccular hair cells.

Depolarization of isolated frog saccular hair cells caused Ca(2+)-dependent increases in membrane capacitance that we interpret as the fusion of synaptic vesicles with the plasma membrane. During a maintained depolarization to -10 mV, the capacitance increased at a rate corresponding to the fusion of approximately 500 vesicles per second at each active zone. Release continued at this high rate for up to 2 s, long enough to exhaust > 5 times the number of vesicles initially in close apposition to the plasma membrane at active zones. We therefore propose that hair cells are specialized for rapid replenishment of vesicles at release sites. Upon repolarization to -70 mV, the capacitance returned exponentially (time constant, approximately 14 s) to near the prestimulus level in perforated-patch recordings, but not in whole-cell recordings, suggesting that a mobile intracellular factor is required for membrane retrieval.

Animals↗
PubMed1994

Calcium signalling in hair cells: multiple roles in a compact cell.

Ca2+ is critical for mechanosensory adaptation, frequency tuning, afferent synaptic transmission, and efferent modulation in hair cells. These four processes involve cytoplasmic Ca2+ in three independent signalling pathways. Recent work suggests that Ca2+ regulates a myosin adaptation motor, and that a mobile Ca2+ buffer is highly concentrated in hair cells. Focal Ca2+ entry and the cytoplasmic Ca2+ buffer help to separate these pathways by limiting the spread of Ca2+ signals.

Animals↗
PubMed1994

Clinical features and treatment outcome for children with CD30+ large-cell non-Hodgkin's lymphoma.

PURPOSE: To determine the frequency of CD30 expression and its relationship to clinical features, immunophenotype, histotype, and outcome in childhood large-cell non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: We reviewed 45 cases of large-cell NHL in children treated at St Jude Children's Research Hospital from 1975 to 1990 for whom there was sufficient tissue to perform immunophenotypic studies. All 45 were screened with a panel of antibodies to detect the presence of CD30 and T-cell and B-cell antigens. Cases were classified according to the National Cancer Institute (NCI) Working Formulation and the Kiel classification system. Clinical features, immunophenotype, pathologic classification, and treatment outcome were compared for CD30+ and CD30- cases. RESULTS: CD30 expression was documented in 18 cases (40%). These 13 boys and five girls had a median age of 13 years at diagnosis. Most (n = 14) had advanced-stage (III and IV) disease. Nodal disease was equally common in CD30+ and CD30- cases, whereas skin involvement was significantly more frequent in CD30+ cases (P = .007). There was no significant association of CD30 expression with histologic subtype according to the NCI Working Formulation, but CD30+ cases were more likely to be anaplastic by the Kiel classification (P < .001). All CD30+ cases had either T-cell or null-cell phenotype, while the majority of CD30- cases were B-cell phenotype (P < .001). Among patients with limited-stage disease, the mean +/- SE estimated 5-year event-free survival (EFS) was 75% +/- 22% for CD30+ cases and 92% +/- 9% for CD30- cases (P = .10); estimates for advanced-stage disease were 57% +/- 17% and 29% +/- 17%, respectively (P = .096). For patients with advanced-stage disease, CD30 expression was associated with a significantly better overall 5-year survival probability (84% +/- 12% v 27% +/- 16%, P = .0016). CONCLUSION: CD30 is frequently expressed in pediatric large-cell NHL and is significantly associated with T-cell or null-cell phenotype, anaplastic morphology, skin involvement, and better overall survival among advanced-stage patients.

Adolescent↗
PubMed1994

Localization of calcium signals by a mobile calcium buffer in frog saccular hair cells.

A recent study (Roberts, 1993) of saccular hair cells from grass frogs (Rana pipiens) has suggested a mechanism by which the unusually high concentrations of calcium-binding proteins found in certain sensory receptors and neurons, particularly in the auditory system, can influence short-range intracellular calcium signaling. In frog saccular hair cells, the mechanism operates within arrays of calcium channels and calcium-activated potassium channels that are involved in the cells' electrical resonance and synaptic transmission. The present study tests the hypothesis that calbindin-D28k, one of the most abundant proteins in these cells, can serve as a mobile calcium buffer that reduces and localizes changes in the intracellular free-calcium concentration ([Ca2+]i) by shuttling calcium away from the channel arrays. Based upon theoretical analysis and computer modeling, it is shown that [Ca2+]i near one or more open channels quickly reaches a steady-state level determined primarily by two properties of the buffer, the mean time (tau c) before it captures a free-calcium ion and a replenishment factor (R), which are related to the buffer's diffusional mobility (DBu), association rate constant (kon), and concentration (Bo) by tau c = (konB0)-1 and R = B0DBu. Simulation of calcium entry through a channel array showed that approximately 1.5 mM of a molecule with the diffusional and binding properties expected for calbindin-D28k (Bo approximately 8 mM calcium-binding sites) is needed to reproduce the previous experimental results. A lower concentration (B0 = 2 mM) was almost completely depleted within the channel array by a modest calcium current (8 pA = 12% of calcium channels open), but still had two important effects: it caused [Ca2+]i to fall steeply with distance outside the array (space constant < 50 nm), and returned [Ca2+]i quickly to the resting level after the channels closed. A high concentration of calbindin-D28k can thus influence the cell's electrical resonance and synaptic transmission. Its most important functions may be to localize regions of high [Ca2+]i and speed the return of [Ca2+]i toward the resting level.

Animals↗
PubMed1993

Tretinoin toxicity in children with acute promyelocytic leukaemia.

Tretinoin is effective in acute promyelocytic leukaemia in adults. Data about its efficacy and safety in children are limited. We have treated 9 children with tretinoin at 45 mg/m2 per day. Pseudotumour cerebri or hyperleucocytosis occurred in 5 patients. Retinoic acid syndrome was seen in 3 cases. 1 of 2 children who developed hyperleucocytosis, pseudotumour cerebri, and retinoic acid syndrome died despite steroids and mechanical ventilation. Complete remissions with tretinoin alone were achieved in 15 patients. All 8 surviving children received consolidation chemotherapy. Our experience with tretinoin therapy suggests that toxicity is frequent in children.

Adolescent↗
PubMed1993

Spatial calcium buffering in saccular hair cells.

The potential importance of intracellular calcium-binding proteins in rapid and highly localized Ca2+ signalling is poorly understood. During fast synaptic transmission, which occurs at specialized active zones where Ca2+ diffuses only a few tens of nanometers from channels to neurotransmitter release sites, a cytoplasmic Ca2+ buffer would have to be extremely fast or present in millimolar concentrations to intercept a significant fraction of the calcium ions en route to their targets. Therefore, Ca2+ buffers have been presumed to be unimportant in fast exocytosis and another fast calcium-mediated process, electrical resonance in hair cells. Here I present evidence to the contrary by showing that hair cells in the frog sacculus contain millimolar concentrations of a mobile cytoplasmic calcium buffer that captures Ca2+ within a few microseconds after it enters through presynaptic Ca2+ channels and carries it away from the point of entry. This spatial buffering reduces the presynaptic free Ca2+ by up to 60 per cent and probably restricts the region in which the internal calcium ion concentration exceeds 1 microM to within < 250 nm of each synaptic site. The buffer can thus influence both electrical resonance and synaptic transmission. Calbindin-D28K or a related protein may serve as the mobile calcium buffer, an action similar to its function in transporting Ca2+ across intestinal epithelial cells.

Animals↗
PubMed1993

Detection of N-myc gene amplification by fluorescence in situ hybridization. Diagnostic utility for neuroblastoma.

We assessed fluorescence in situ hybridization (FISH) as an alternative to Southern blot analysis for determination of N-myc gene amplification in neuroblastoma. In the 44 pediatric solid tumor cell lines examined (20 neuroblastomas), the mean number of N-myc copies determined by FISH correlated closely with Southern blot results. There was wide intercellular variability in gene copy number in tumors that had evidence of amplification; however, tumors judged to be non-amplified completely lacked any cells with high N-myc copy number. FISH provided reliable estimates of N-myc amplification in 12 clinical samples even when the percentage of tumor was low. The other advantages of FISH over Southern blot analysis were speed and technical simplicity, ability to discern heterogeneous gene amplification among tumor cells in the same specimen, and capacity to determine the source of the amplified N-myc signal, whether extrachromosomal double-minute chromosomes, expanded intrachromosomal regions, or chromosome 2 aneuploidy. We conclude that FISH would refine the analysis of N-myc amplification in neuroblastoma and thus improve the assignment of patients to prognostic groups based on this unfavorable risk factor.

Blotting, Southern↗
PubMed1992

Fusion of the leucine zipper gene HLF to the E2A gene in human acute B-lineage leukemia.

A t(17;19) chromosomal translocation in early B-lineage acute leukemia was shown to result in chimeric transcripts that contain sequences from the E2A basic helix-loop-helix transcription factor gene on chromosome 19, fused to sequences from a previously unidentified gene (HLF) on chromosome 17 that encodes a hepatic leukemia factor. The chimeric protein consisted of the amino-terminal transactivation domain of E2A linked to the carboxyl-terminal basic region-leucine zipper domain of HLF. HLF was normally expressed in liver and kidney, but not in lymphoid cells, and was found to be closely related to the leucine zipper-containing transcription factors DBP (albumin D-box binding protein) and TEF (thyrotroph embryonic factor), which regulate developmental stage-specific gene expression.

Adenovirus Early Proteins↗
PubMed1992

Transcription of the human colony-stimulating factor-1 receptor gene is regulated by separate tissue-specific promoters.

Receptors for macrophage colony-stimulating factor (CSF-1R) are expressed not only by monocytes, macrophages, and their progenitors, but also by placental trophoblasts during fetal development. In monocytes, CSF-1R gene transcripts originate at multiple sites immediately upstream of the gene's coding sequences, whereas in placental cells the transcripts include an additional noncoding exon, located 26 kb upstream near the 3' end of the B-type platelet-derived growth factor (PDGF) receptor gene. Physically distinct CSF-1R transcription origins suggest separate promoter usage by the two cell types. To identify regulatory elements of these promoters, we fused CSF-1R genomic sequences to bacterial reporter genes and introduced the resulting constructs into human cell lines and mouse fibroblasts. A 775-bp genomic fragment containing CSF-1R placental transcription origins and adjacent upstream sequences mediated reporter gene expression in BeWo choriocarcinoma cells and mouse NIH-3T3 fibroblasts, but not in myeloid or lymphoid cells. By contrast, a 550-bp genomic fragment containing CSF-1R monocyte transcription origins and 5' flanking sequences directed gene expression in U-937 human myeloid cells, but not in the other cell types. Thus, nucleotide sequences of fewer than 1,000-bp upstream of the two independent CSF-1R transcription origins contain the minimal promoter elements needed to program appropriate tissue-specific expression of reporter genes.

Base Sequence↗
PubMed1992

Sublocalization of the chromosome 5 breakpoint of the 3;5 translocation in myelodysplastic syndromes and acute myeloid leukemia.

A t(3;5)(q25.1;q34) reciprocal translocation identifies a subset of cases of myelodysplastic syndrome or acute myeloid leukemia (AML) that are characterized by increased numbers of megakaryocytes and severe trilineage dysplasia. As a first step in characterizing the t(3;5) breakpoints, we asked whether the translocation involves the CSFIR/PDGFRB locus at 5q33-q35. Pulsed-field gel electrophoretic analysis of a region extending 580 kb 5' to the PDGFRB gene and 120 kb 3' to the CSFIR gene did not reveal aberrant restriction fragments in leukemic cell DNA, confirming that the breakpoint does not occur in the vicinity of these genes. To sublocalize the breakpoint, we performed Southern blot hybridizations using DNA from human x hamster somatic cell hybrids containing the normal 3, the normal 5, the derivative 3, or the derivative 5 human chromosome. Using a series of polymorphic DNA probes from the long arm of chromosome 5, which have been linked by genetic recombination, we bracketed the breakpoint to within a region that spans approximately 13 centimorgans (sex average) and is flanked by the q34-qter markers cKK5.19 and L1200 (D5S62). This analysis places the chromosome 5 breakpoint of the t(3;5) considerably telomeric to the CSFIR/PDGFRB locus, confirming our studies with pulsed-field electrophoresis. Future efforts to identify the genes affected by the t(3;5) should focus on the 5q segment described in this study.

Animals↗
PubMed1992

Hormonal regulation and segmental specificity of motoneuron phenotype during metamorphosis of the tobacco hornworm, Manduca sexta.

The abdominal prolegs of Manduca sexta larvae are eliminated at the onset of metamorphosis. Previous work showed that the prepupal peak of ecdysteroids in the hemolymph causes the dendritic arbors of proleg motoneurons to regress and a stereotyped subset of the motoneurons to die. In the present study we investigated the parameters of ecdysteroid exposure that are important for eliciting these responses by directly infusing 20-hydroxyecdysone (20-HE) into the hemolymph of insects deprived of their own endocrine glands. Doses of 20-HE that were near threshold for evoking regression or death were consistently more effective when infused over a longer duration. Theoretical calculations of hemolymph hormone profiles produced by the infusions support a model of ecdysteroid action in which the hormone concentration must remain above a threshold level for a critical duration of time to be physiologically effective. We further found that segmental location can influence both the metamorphic fate and the hormonal sensitivity of Manduca motoneurons.

Animals↗