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Biomedical subjects

J V Watson

Publications and source records attributed to J V Watson.

At least 73 records · Page 4Linked to original sources
PubMed1987

Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system.

We have used a novel approach to investigate the control of initiation of replication of sperm nuclei in a Xenopus cell-free extract. Nascent DNA was labelled with biotin by supplementing the extract with biotin-11-dUTP, and isolated nuclei were then probed with fluorescein-conjugated streptavidin. Flow cytometry was used to measure the biotin content of individual nuclei and their total DNA content. This showed that incorporation of the biotinylated precursor increases linearly with DNA content. Haploid sperm nuclei replicate fully to reach the diploid DNA content over 2-6 h in the extract. Synthesis stops once the diploid DNA content is reached. Different nuclei enter S phase at different times over greater than 1.5 h, although they share the same cytoplasmic environment. Nuclei reach their maximum rates of synthesis soon after entry into S phase and some replicate fully in less than 0.5 h, resembling the rates of replication observed in the intact egg. These results indicate that initiations are coordinated within each nucleus such that the nucleus is the fundamental unit of replication in the cell-free system.

Animals↗
PubMed1987

Oncogene expression in ovarian cancer: a pilot study of c-myc oncoprotein in serous papillary ovarian cancer.

The nuclear-associated protein product of the c-myc gene, p62c-myc, was assayed simultaneously with total DNA using flow cytometry in nuclei extracted from archival biopsies of serous papillary carcinoma of the ovary. The oncoprotein was probed with a synthetic peptide-induced mouse monoclonal antibody which was subsequently labeled with a fluorescent rabbit anti-mouse immunoglobulin and DNA was assayed using the nucleic acid fluorochrome propidium iodide. Serous papillary ovarian carcinoma expressed significantly higher p62c-myc levels compared with normal ovary (P less than 0.00003 Mann-Whitney U test). Biopsies classified as "borderline" low-potential malignancy exhibited levels between normal ovary and carcinoma. The difference between normal and "borderline" was significant at P less than 0.003, but no difference between "borderline" and frankly invasive biopsies was observed, P = 0.149. There was no difference among the histological grades of carcinomas. All normal ovaries had diploid DNA content as did 5/6 cases of "borderline" malignancy. The majority of cases of carcinoma, 28/36, were aneuploid. There was a statistically significant difference in the distribution of aneuploidy, P less than 0.005, between invasive carcinomas and those classified as "borderline" low-potential malignancy.

Aneuploidy↗
PubMed1987

Flow cytometric quantitation of the c-myc oncoprotein in archival neoplastic biopsies of the colon.

The c-myc oncogene encoded protein product, p62c-myc, was assayed simultaneously with DNA in populations of individual nuclei extracted from archival biopsies of colonic neoplasia. Both the protein and DNA were assayed fluorimetrically using flow cytometry with a synthetic peptide induced monoclonal antibody (MYC 1-6E10) for the protein and propidium iodide for DNA. The nuclear p62c-myc levels increased progressively from normal mucosa through polyps to carcinomas. However, there was a trend for the more poorly differentiated carcinomas to exhibit lower levels than moderately and well-differentiated tumours, p = 0.085. These results agree with those published previously with the same antibody using Western blotting for protein extracted from fresh frozen tissue and immunocytochemical assessment. Furthermore, flow cytometry is able to effect discriminations between subsets in heterogeneous populations using DNA as a second parameter which Western blot bulk studies cannot.

Biopsy↗
PubMed1987

New methods for implanting plastic tubes for 192iridium brachytherapy using steel needles.

A revised method for implanting plastic tubes used to carry 192iridium for brachytherapy is described. Solid malleable stainless steel needles are machined at one end with a tapered screw thread, on to which the plastic tubing can be placed for pulling through tissue. The technique has proved to be both simpler and quicker than the standard 'Amersham' method. Since the solid needles may be moulded, it is easier to implant relatively inaccessible areas such as axillary, intrathoracic and paraspinal sites. For sites with no clear exit point for the needles, a large diameter steel needle is described, which enables the plastic tubes to be inserted with less trauma to the tissues.

Brachytherapy↗
PubMed1987

Flow cytometric quantitation of DNA and c-myc oncoprotein in archival biopsies of uterine cervix neoplasia.

The c-myc nuclear associated oncoprotein has been quantitated simultaneously with DNA in nuclei extracted from archival biopsies of uterine cervix neoplasia. The oncoprotein and DNA were measured fluorimetrically in a flow cytometer using a mouse monoclonal antibody (MYC 1-6E10) and propidium iodide. Normal biopsies exhibited higher oncoprotein levels than carcinomas (P less than 0.00001). Furthermore, the maximum fluorescence signal in the normal tissue occurred at a lower antibody concentration compared with tumour tissue. There was no correlation between oncoprotein levels and histological grade, stage of disease, age of the patients or prognosis in the carcinomas. Aneuploidy, defined as a distinct second peak separate from the diploid distribution, was not a significant feature. The c-myc oncoprotein nuclear content does not appear to be a prognostic indicator in carcinoma of the cervix from the results of these studies but there is clearly diagnostic potential, particularly for automated analysis of cervical screening.

Cervix Uteri↗
PubMed1987

Abnormal distribution of c-myc oncogene product in familial adenomatous polyposis.

Monoclonal antibodies raised by synthetic peptide immunisation were used to determine the distribution of the protein product of the c-myc gene by immunocytochemical staining of archival wax embedded material from patients with familial adenomatous polyposis. Polyps from 18 cases of familial adenomatous polyposis, 10 of whom had developed malignant change, and 30 normal control colonic biopsy specimens were examined. A consistent staining pattern was observed in normal mucosa; nuclear staining in the basal proliferative zone; mixed nuclear and cytoplasmic staining in the maturation zone; and cytoplasmic localisation in the surface mature zone. In contrast, the polyps and carcinomata showed a mixed pattern of cytoplasmic and nuclear localisation in the basal proliferative zone with nuclear persistence throughout the crypts to the surface mature zone. This abnormal distribution of the c-myc oncogene product may have a role in the evolution of polyps and their subsequent malignant transformation into familial adenomatous polyposis.

Adenomatous Polyposis Coli↗
PubMed1986

Molecular calibration in flow cytometry with sub-attogram detection limit.

A monoclonal antibody to complement receptor type 1 (CR1) has been double labelled with 125iodine and fluorescein isothiocyanate (FITC) and used for parallel radioimmune assay (RIA) and flow cytometric studies. Serial dilutions of the reagent with unlabelled antibody were used to obtain a linear correlation between radioactivity counts and fluorescence over the whole range of labelled antibody molecules bound per cell, namely 700-25,000. This has enabled us to calibrate our flow cytometer in terms of numbers of molecules per channel as opposed to the customary arbitrary fluorescence units. Normal human granulocytes from a single donor were found to have a mean of 25,000 CR1 molecules per cell with a normal distribution and standard deviation of 4775 molecules. The detection limit of the flow cytometer was 730 CR1 molecules per cell which is equivalent to between 125 and 150 molecules of free fluorescein. This represents less than 0.1 attogram (ag) of fluorescein per cell, where 1 ag = 10(-18) g. The resolution was 44 CR1 molecules per digitization step of the analogue-to-digital converters (ADC) which corresponds to the fluorescence from less than ten molecules of free fluorescein.

Calibration↗
PubMed1986

Estimating the kinetic parameters for enzymatic drug metabolism in the whole animal.

A method for estimating the Michaelis constant, Km, and the maximum reaction velocity, Vmax, for the enzymatic degradation of a parent compound to a metabolite in the intact animal is presented. The technique involves a mathematical analysis which has shown that under specific conditions the peak/plateau blood concentrations of metabolite are related to initial parent compound concentration by the Michaelis-Menten relationship. It has also been shown how these data can be analysed with the "direct linear plot" of Eisenthal and Cornish-Bowden (Biochem. J. 139, 715 (1974)) to yield the enzyme kinetic parameters.

Animals↗
PubMed1986

The application of flow cytophotometry in measurements of cell adhesion.

A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These "free" cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer. A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from inaccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%-10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Colcemid inhibits growth during early G1 in normal but not in tumorigenic lymphocytes.

Mitogenically stimulated human and mouse lymphocytes enter the cell cycle (G0, G1A, G1B, S, G2+M) via a newly recognized subphase, G1'. This subphase precedes G1A and is distinct from G0. The G1' subphase is absent in immortalized and tumorigenic lymphoblastoid cell lines (LCLs) by cytofluorimetric criteria. Furthermore, colcemid inhibits transition through the G0/G1' as well as G2 phases in mitogen-stimulated lymphocytes and in LCLs. Tumorigenic LCLs are not sensitive to growth inhibition by colcemid during early G1. These observations suggest that a progressive series of changes have occurred during G0/G1' which lead to deregulation of growth control.

Animals↗
PubMed1986

The clinical significance of flow cytometric c-myc oncoprotein quantitation in testicular cancer.

A sensitive flow cytometric assay has been developed using a monoclonal antibody, Myc 1-6E10, to quantitate c-myc oncoprotein levels in nuclei isolated from wax embedded testicular tumours. The oncoprotein (p62c-myc) level increased significantly with increasing teratoma differentiation. Patients with intermediate and undifferentiated tumours who developed recurrence had lower p62c-myc levels than those who were disease free since their initial treatment. Such quantitative biochemical methods may provide new prognostic indices for cancer patients.

Antibodies, Monoclonal↗
PubMed1986

Neuron specific enolase expression in carcinoma of the lung.

The value of neuron specific enolase (NSE) immunoreactivity as a marker for small cell lung cancer (SLC) has been assessed using a monoclonal antibody (MCAB) against NSE, MCAB specificity was confirmed using purified enolase isoenzymes, sections of human brain, a panel of lung tumours, neuroendocrine and non-neuroendocrine tumours and normal tissues. Using this MCAB in radioimmunoassay and immunohistochemistry, NSE immunoreactivity was detected in all SCLC material examined. However, considerable reactivity was also observed in a number of non-small cell lung cancer cell lines and tumour biopsy specimens. Furthermore, intratumoral heterogeneity with respect to NSE immunostaining was observed in several cases. Factors which may underlie such intratumoral phenotypic diversity were assessed using flow cytometry together with MCABs directed against both NSE and non-neuronal enolase. Such studies revealed that enolase expression in cells which were no longer actively proliferating differed markedly from that of cells in exponential growth. Furthermore, cells grown under conditions of increasing hypoxia exhibited increased enolase expression relative to those grown under oxygenated conditions. It is concluded from these studies that NSE immunoreactivity per se is an unreliable marker for the SCLC phenotype.

Animals↗
PubMed1986

Predominant role for DNA damage in etoposide-induced cytotoxicity and cell cycle perturbation in human SV40-transformed fibroblasts.

The cytotoxic and cell kinetic effects of the epipodophyllotoxin 4,6-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyr anoside) (VP-16) in cultured mammalian cells are thought to relate to the induction of DNA damage, specifically DNA strand interruptions. In an effort to explore this relationship in human cells we have identified a VP-16-hypersensitive human cell system, namely an SV40-transformed fibroblast line (AT5BIVA) originally derived from an ataxia telangiectasia (AT) patient. Evidence is presented that enhanced VP-16 sensitivity may be a consistent in vitro feature at AT derived cells. However, the intrinsic sensitivity (DNA strand breaks per lethal hit quantitated by nucleoid sedimentation) was the same for AT5BIVA and a corresponding normal control, indicating that the AT cell line accumulated more drug-induced DNA damage during short-term VP-16 exposures. It is suggested that AT cells may have abnormal topoisomerase II activity. The cell cycle responses of normal and AT cells to VP-16 exposure were complex, with the generation of parasynchronous S phase populations and the accumulation of cells in G2. Differences in cell killing or DNA strand breakage between normal and AT cells could only be correlated with the magnitude and kinetics of the G2 retention phenomenon. In short, there are several similarities in the action of ionizing radiation and VP-16. We suggest that the sensitivity of cellular DNA to VP-16-induced DNA damage and the kinetics of the G2 delay may be useful parameters for predicting the survival probability of drug-treated human tumor populations.

Ataxia Telangiectasia↗
PubMed1985

A simultaneous flow cytometric assay for c-myc oncoprotein and DNA in nuclei from paraffin embedded material.

A simultaneous flow cytometric assay for the c-myc oncoprotein and DNA in nuclei extracted from archival paraffin wax embedded clinical biopsies is presented. The nuclei were extracted by pepsin digestion after dewaxing 20 micron sections. The c-myc oncoprotein was probed with a mouse monoclonal antibody. This was raised against a synthetic peptide corresponding to a hydrophilic region of the protein predicted from the amino acid sequence. The technique is illustrated with biopsies from patients with testicular cancer and with benign and malignant neoplasms of the colon.

Antibodies, Monoclonal↗