Search PubMed⌕ Search

Biomedical subjects

B W Stewart

Publications and source records attributed to B W Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1993

Etoposide-induced cytotoxicity in two human T-cell leukemic lines: delayed loss of membrane permeability rather than DNA fragmentation as an indicator of programmed cell death.

Features of the apoptotic response evident in glucocorticoid-treated thymocytes are not uniformly observed in cell lines exposed to anticancer drugs. The significance of such variation has been assessed by monitoring molecular and cellular processes induced by etoposide (VP-16) in the human lymphoblastoid T-cell lines CCRF-CEM (CEM) and MOLT-4 contrasted, where appropriate, with those induced by necrotizing injury. Cytotoxic concentrations of the drug were determined to be 5-100 microM on the basis of tetrazolium reduction assay. The two lines were equally sensitive to VP-16; no difference in concentration of drug which inhibited cell growth by 50% with respect to control (i.e., drug free) cultures was apparent irrespective of exposure times from 3-72 h. DNA strand breaks were evident in both populations within 3 h of exposure to VP-16. Morphological change, assessed microscopically, involving nuclear condensation and cell shrinkage was qualitatively and quantitatively similar in VP-16-treated CEM and MOLT-4 cells. Flow cytometric analysis indicated that the G2/M fraction of the randomly dividing MOLT-4 population was approximately one-third that of CEM cells, but each line exhibited a decrease in this fraction 3-6 h after treatment. Despite these similarities, marked differences in the response to VP-16 were evident in the two populations. Internucleosomal fragmentation, detected electrophoretically 3 h after treatment in DNA isolated from CEM cells, was not detected under any condition in MOLT-4 DNA. Apoptotic bodies, also evident within 3 h of VP-16 treatment of CEM cells, were not readily apparent in MOLT-4 cells under the same conditions. Treatment causing necrosis resulted in trypan blue uptake within 1 h in a similar high proportion of cells from both lines. Exposure to VP-16 resulted in such a loss of membrane integrity by 6 h in CEM cells, while change in this parameter occurred only after 24 h in the case of MOLT-4 cells. The findings indicate a wide scope of difference in apoptotic response and suggest delayed loss of membrane permeability, rather than DNA fragmentation, as the clearest indicator of programmed cell death in these populations.

Apoptosis↗
PubMed1993

Determination of N-myc gene amplification in neuroblastoma by differential polymerase chain reaction.

Determination of N-myc gene amplification, a powerful prognostic indicator in the childhood tumour, neuroblastoma, has routinely been performed by Southern analysis. We have developed a differential polymerase chain reaction (PCR) assay, in which the N-myc target gene is co-amplified with a control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Following electrophoresis, a ratio between the two PCR products within a given DNA sample is then determined by densitometry. This assay was applied to DNA isolated from 32 primary neuroblastoma tumours for which the N-myc status had previously been determined by Southern analysis. Following PCR, samples containing a single copy of the N-myc oncogene were clearly distinguishable from samples with N-myc gene amplification, based on an N-myc/GAPDH ratio of below or above 1.0, respectively. Linear regression indicated a highly significant relationship (R = 0.94; P < 0.0001) between N-myc copy number (Southern) and N-myc/GAPDH ratio (PCR). Serial dilution of N-myc amplified DNA with non-amplified control DNA indicated that the PCR assay was sufficiently sensitive to detect two-fold amplification. Moreover, such serial dilution allowed determination of N-myc copy number. The assay, which requires only small amounts of tissue and does not utilize 32P-radioactivity, therefore provides a rapid and sensitive alternative to Southern analysis.

Base Sequence↗
PubMed1993

Karyotypic analysis of CCRF-CEM and drug-resistant cell lines with stable and unstable ploidy.

Karyotypic studies were performed on methotrexate (MTX)- and vincristine (VCR)-resistant cell lines derived from the human T-cell leukemia CCRF-CEM cell line. We noted karyotypic selection and additional chromosome change associated with acquisition of drug-resistance. Furthermore, we observed that both the parental and MTX-resistant sublines, CEM/MTX R1-3, had a tendency to ploidy change. Karyotypic studies of malignant cells have shown that polyploidy is frequently a consequence of a single sporadic event, followed by growth and selection of the polyploid clone [1]. In this study, however, various karyotypic clones were identified with near- and pseudotetraploid karyotypes that appeared to be derived from different near- and pseudodiploid sidelines. Polyploidy was invariably associated with loss of at least two of the four chromosomes 8 whether the pseudodiploid sideline from which it was derived carried one or both chromosomes 8. In contrast, neither polyploid clones nor loss of chromosome 8 was noted in the CEM/VCR R cells.

Chromosome Aberrations↗
PubMed1992

Association of N-myc amplification with neuroblastoma: the Australian and New Zealand experience.

Tumour samples from 38 patients with neuroblastoma were analysed for the presence of N-myc amplification. N-myc gene copy number in tumour DNA was determined by Southern blotting, and by dilution analysis where appropriate. Available clinical data, obtained at tissue collection and by subsequent questionnaire included patient age at diagnosis, catecholamine, ferritin and neuron-specific enolase levels, treatment and disease status. This study was designed to investigate the use of N-myc amplification data as an additional indicator for determination of prognosis. Patients with amplified N-myc had more rapid disease progression than those without amplification (P less than 0.005). Stratification of Stage III and IV patients using N-myc amplification permitted identification of a subgroup with poorer prognosis. The results demonstrate that determination of N-myc amplification is important in assessment of prognosis and subsequent treatment in patients with neuroblastoma.

Abdominal Neoplasms↗
PubMed1992

Effect of an arginine-glycine-aspartic acid-containing peptide on hematogenous candidal infections in rabbits.

The adherence of Candida albicans yeast cells to the subendothelial extracellular matrix, fibronectin, laminin, and type I and IV collagen was tested. Fibronectin (10(-7) M) and a peptide, PepTite-2000 (Telios Pharmaceuticals Inc., San Diego, Calif.), containing the sequence arginine-glycine-aspartic acid (RGD) inhibited Candida adherence to these targets by greater than 90%. When C. albicans was perfused over ex vivo rabbit aortic endothelium, there was no significant difference in the amount of adherence in the presence or absence of the RGD-containing peptide. However, the RGD-containing peptide reduced the number of Candida organisms present in liver, brain, heart, and kidneys (P less than 0.05) of rabbits 4 h after intravenous inoculation of 5 x 10(7) C. albicans yeast cells. The peptide also reduced the number of macroscopic Candida abscesses in the kidneys of rabbits 72 h after intravenous inoculation of 10(7) C. albicans yeast cells (P less than 0.05). Inhibition of Candida adherence in vitro and in vivo may occur because the peptide blocks a fungal receptor that is necessary for adherence.

Animals↗
PubMed1991

Reduced drug accumulation as the mechanism of extreme clinical resistance to methotrexate in the human T-cell leukemia xenograft, LALW-2.

The mechanisms were examined that underlie the extreme resistance to methotrexate (MTX) by near diploid leukemic T-cells (LALW-2) exposed to the drug only during the course of therapy administered to the patient of origin. Despite the LALW-2 cells being highly resistant to MTX (inhibitory dose for 50% of cells, more than 10(-3) mol/l), southern blot analysis did not show any amplification of the dihydrofolate reductase gene, nor was there any evidence, by comparison with drug-sensitive CCRF-CEM cells, that the gene was overexpressed. Kinetic analysis of dihydrofolate reductase activity in the presence of MTX provided no basis for attributing resistance in LALW-2 cells to a change in enzyme structure. By contrast, studies of MTX accumulation revealed that the LALW-2 cells accumulated significantly less drug than either CCRF-CEM cells or a MTX-resistant CCRF-CEM subline with a characterized transport defect. These data suggest that extreme MTX resistance in LALW-2 cells is mediated by reduced drug accumulation in the absence of any effect on the target enzyme.

Animals↗
PubMed1991

Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells.

In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to [3H]thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

Caffeine↗
PubMed1990

Phenotypic and cytogenetic analysis of atypical multidrug resistance in human leukaemic cells selected with methotrexate at high concentration.

A series of CCRF-CEM sublines selected for extreme resistance to methotrexate has been shown previously to exhibit cross resistance to a number of agents belonging to the multidrug resistance phenotype. The mechanism(s) underlying resistance to vincristine, vinblastine and actinomycin D in the most resistant subline (CEM/MTX R3) has now been investigated. Efflux of [3H]vincristine was more rapid in CEM/MTX R3 than in either CCRF-CEM cells or a methotrexate-resistant subline not refractory to Vinca alkaloids. In addition, verapamil completely reversed resistance to vincristine, vinblastine and actinomycin D in the CEM/MTX R3 cells. While these results are suggestive of P-glycoprotein-mediated multidrug resistance, Northern analysis revealed no detectable expression of the mdr 1/gene in CEM/MTX R3 cells. Likewise, karyotypic analysis of the resistant subline, while revealing certain clonal abnormalities, provided no evidence of alteration in the mdr 1/gene locus on chromosome 7. The data suggest therefore the operation, in these cells, of a novel mechanism of resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Heterotransplantation of human lymphoid neoplasms using a nude mouse intraocular xenograft model.

There are few effective models for the study of human lymphoid neoplasms, including in vivo xenografts in immunocompromised animals. Exploiting the additional immune privilege of the anterior chamber of the nude mouse eye, a novel method of direct heterotransplantation of cells from childhood leukemias and lymphomas has been developed. The establishment and characterization of 18 lymphoid xenograft cell lines maintained in the nude mouse intraocular model are reported. Cell sources for heterotransplantation were specimens of bone marrow, peripheral blood, or lymphomatous masses obtained at either diagnosis or recurrence of disease in the patients. The 18 patients and resultant cell lines were grouped into four immunophenotypic categories: Category 1, B-lineage (pre-B and early pre-B), "common" acute lymphatic leukemias; Category 2, cell lines of similar immunophenotype derived from patients with unusual features; Category 3, B-cell neoplasms and cell lines; and Category 4, neoplasms and cell lines in part or totally of T-cell origin. With reference to these groupings, rates of ingraftment from clinical specimens varied according to immunophenotype and disease status: Category 1, 1 of 15 at diagnosis, 5 of 7 at relapse; Category 2, 1 of 1 at diagnosis, 2 of 2 at relapse; Category 3, 6 of 6 at diagnosis; and Category 4, 2 of 9 at diagnosis, 1 of 1 with persistent disease. Rearrangements of the genes for immunoglobulin heavy chain or kappa light chain and for beta subunit of the T-cell receptor gene were demonstrated according to immunophenotype, with the exception of one cell line which showed no rearrangements. Evidence of Epstein-Barr virus DNA was shown in only one cell line, of B-cell immunophenotype. Cytology, histopathology, and electron microscopy in representative patient and xenograft samples demonstrated correlations between the specimens of origin and cells or sections from ingrafted tumors in mice. It is concluded that the direct heterotransplantation of cells from childhood leukemias and lymphomas to the anterior chamber of the nude mouse eye provides a relevant and reproducible model for the maintenance and study of human lymphoid neoplasms.

Animals↗
PubMed1990

Structural features of the amplified N-myc oncogene detected by micrococcal nuclease digestion of neuroblastoma cell nuclei.

The structure of chromatin containing amplified N-myc in neuroblastoma and retinoblastoma cells was investigated using micrococcal nuclease digestion of isolated nuclei. The size distribution of DNA fragments containing N-myc, produced by micrococcal nuclease digestion of nuclei, was determined and compared to that of DNA containing the structural gene for dihydrofolate reductase. A perturbation of the native structure of chromatin containing N-myc was evident from the association of N-myc with more extensively digested DNA when compared with chromatin containing dihydrofolate reductase.

Cell Nucleus↗
PubMed1990

Structural analysis of replicating DNA following exposure to cytotoxic drugs: implications for current models of DNA synthesis in mammalian cells.

The polynucleotide length of single-stranded regions in double-stranded DNA may be determined by caffeine gradient elution from benzoylated DEAE-cellulose. On the basis of this principle, analysis has been made of sheared, deproteinized DNA isolated from synchronized lymphoblastoid cells. Two classes of single-stranded regions were detected. A minor fraction of replicating DNA contained single-stranded regions of 200 nucleotides, whilst the major structural discontinuity involved single-stranded regions of 1-4 kilobases. Newly incorporated [3H]thymidine was principally associated with the latter. Using a 'pulse-chase' protocol, the effect of certain cytotoxic drugs (and related compounds) on the proportion of replicating DNA exhibiting single-stranded character was assessed. The effects were variable. The proportion was increased by hydroxyurea and 3-aminobenzamide, but decreased by inhibitors of DNA polymerase and, to a greater extent, by inhibitors of topoisomerase. Caffeine gradient elution associated drug-induced changes with the radiolabelling of long single-stranded regions. The results are consistent with models of DNA replication involving DNA polymerization remote from replicating forks.

Antineoplastic Agents↗
PubMed1989

Atypical multidrug resistance in a therapy-induced drug-resistant human leukemia cell line (LALW-2): resistance to Vinca alkaloids independent of P-glycoprotein.

Near diploid leukemic T-cells (LALW-2), exposed to cytotoxic drugs only as a consequence of therapy administered to the donor patient, have been maintained by serial xenograft in nude mice. In comparison with the leukemic line CCRF-CEM, using a growth inhibition assay, LALW-2 cells were resistant to Vinca alkaloids and actinomycin D (relative resistance, 200-fold or more), were slightly resistant to Adriamycin (relative resistance, 4-fold), and showed no resistance to daunorubicin or teniposide. By comparison, a vincristine-resistant CEM subline developed in our laboratory (CEM/VCR R) was resistant to all these agents by at least 30-fold. The VCR R subline served as a positive control, confirming the previously reported correlation between multidrug resistance and amplification of the P-glycoprotein gene. Comparison of CEM, CEM/VCR R, and LALW-2 cells establish that the P-glycoprotein gene was not amplified or overexpressed in the LALW-2 cells; neither could the gene product be detected by immunoblotting in extracts from these cells. The LALW-2 cells were further distinguished from CEM/VCR R cells due to the lack of increased vincristine efflux by the xenografted cells, an effect readily demonstrable in the CEM/VCR R cells. However, although LALW-2 cells efflux vincristine at the same rate as CCRF-CEM cells, the xenografted cells exhibited a reduced rate of vincristine accumulation. Uptake of daunorubicin by LALW-2 cells was not distinguished from that by CEM cells, consistent with similar 50% inhibitory dose levels for this drug in both cell populations, and differentiating both from CEM/VCR R cells. Thus, clinical resistance in this case appears to be an "atypical" form of multidrug resistance specifically distinguished by resistance to Vinca alkaloids and actinomycin D occurring in the absence of increased amounts of P-glycoprotein and manifesting decreased drug uptake.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Resistance to drugs associated with the multidrug resistance phenotype following selection with high-concentration methotrexate.

To study patterns of resistance at extreme but nevertheless clinically relevant drug concentrations, we developed a series of methotrexate-selected CCRF-CEM sublines, all of which were highly resistant to this antifolate (relative resistance, 10(2)- to greater than 10(5)-fold). The least methotrexate-resistant subline was completely sensitive to drugs associated with the multidrug resistance phenotype. However, more highly methotrexate-resistant sublines were significantly cross-resistant to vincristine, vinblastine, and dactinomycin (maximum relative resistance, 40-fold). These sublines were not cross-resistant to doxorubicin, daunorubicin, and teniposide. Regression analysis indicated that relative resistance to methotrexate was correlated with relative resistance to vincristine (r = 0.96) and vinblastine (r = 0.99). Such cross-resistance in highly methotrexate-resistant cells may have important clinical implications.

Antineoplastic Agents↗
PubMed1989

Therapy-induced drug resistance in a human leukemia line (LALW-2). A clinically relevant model.

A human leukemic T-cell line, LALW-2, established by xenografting in nude mice, has been maintained through 14 serial passages. The cells display consistent morphologic features, immunophenotype, and karyotypic aberrations (including an 11;14 translocation) and exhibit rearrangement of the T-cell receptor beta-chain gene. The growth rate of LALW-2 xenografts was differentially affected by drugs administered to host mice, the cells being resistant to cytotoxic agents (particularly methotrexate and doxorubicin) used in treatment of the donor patient. In short-term in vitro culture, LALW-2 cells exhibited extreme resistance to methotrexate and were also resistant to vincristine, vinblastine, dactinomycin, and doxorubicin. The findings differ from those obtained with laboratory-derived methotrexate or multidrug-resistant cell lines. The response of LALW-2 cells, in both the nude mouse model and in vitro, is consistent with acquisition of drug-resistance as a result of clinical treatment.

Animals↗
PubMed1989

Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: preferential solubilization of nascent RNA at low enzyme concentration.

1. Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments. 2. Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some greater than 22 kb long. 3. Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction. 4. We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase.

Animals↗
PubMed1988

Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography.

The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.

Bacteriophage phi X 174↗