Biomedical subjects

G A Dent

Publications and source records attributed to G A Dent.

26 records · Page 2Linked to original sources

PubMed1989

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content.

Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 micrograms/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 micrograms lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein isothiocyanate conjugated (FITC) antimyeloperoxidase (an azurophil granule enzyme), or with an anti-c-myc antibody and FITC goat anti-mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.

Antigens, Surface↗

PubMed1989

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide.

The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G1/G0 and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G1 DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G1 to G0.

Blotting, Northern↗

PubMed1988

Leukemic cell maturation: phenotypic variability and oncogene expression in HL60 cells: a review.

Leukemic cells in vitro and in vivo demonstrate an unstable phenotype. We have observed in HL60 cells maintained in culture over long periods of time such phenomena as emergence of drug resistance, oncogene amplification, loss of granulocyte and monocyte lineage markers, and alteration in cell growth parameters. As summarized in this report, a comprehensive review of the literature on HL60 cells shows a wide diversity of phenotype for these cells. We have developed and acquired from other laboratories a series of HL60 sublines with varying phenotypic characteristics. The time in continuous log phase culture for HL60 cells (passage 13 ATCC, Rockville MD) to undergo phenotypic drift from a heavily granulated promyelocytic cell to a more undifferentiated agranular blast form on four occasions varied from 3 to 18 months. The actual loss of promyelocytic phenotype occurred rapidly (within less than 1 month) following a variable period of apparent stable phenotype, The change in morphology was invariably accompanied by decreased sensitivity to ARA-c (5- to 20-fold increase in LD50 and dose necessary to induce NSE positive cells). The c-myc gene is variably amplified in sublines of HL60 cells. The expression of c-myc primarily reflects alterations in cell cycle kinetics and was not found to be correlated with a switch between proliferation and maturation. These results suggest that phenotypic drift may be due to loss of response to regulatory signals that affect the expression of a number of cellular genes.

Cell Differentiation↗

PubMed1987

Estrogen receptor determination by monoclonal antibody in fine needle aspiration breast cancer cytologies: a marker of hormone response.

A monoclonal antibody (H222) prepared against purified estrogen receptor has been demonstrated to be highly specific and sensitive in an immunohistochemical assay for the detection and quantification of estrogen receptor in human breast carcinoma biopsy specimens. To evaluate the clinical applicability of this assay in cytologic specimens, response to therapy in 12 patients with recurrent breast cancer was correlated with H222 antibody localization in specimens obtained by fine needle aspiration biopsy. All 7 patients with positive H222 responded to hormone therapy (1 complete and 3 partial remissions and 3 stable disease), while 1 of 5 patients with negative H222 responded (1 stable disease). This experience suggests a role for immunohistochemical estrogen receptor determinations in prediction of response to hormonal therapy in patients whose tumor is accessible only to aspiration biopsy. Special care in the handling and interpretation of specimens is essential to optimal application of this method.

Adult↗

PubMed1986

Use of monoclonal antiestrogen receptor antibody to evaluate estrogen receptor content in fine needle aspiration breast biopsies.

A monoclonal antibody prepared against estrogen receptor has been shown to be highly specific and sensitive for the detection and quantification of estrogen receptor in human breast lesions using immunohistochemical methods. A semiquantitative relationship has been shown between the intensity of staining and biochemical receptor analysis. To evaluate the usefulness of this technique in fine needle aspiration biopsies, 41 cases of breast cancer were studied. Nuclear localization of receptor antibody PAP complex was observed. Comparison of the immunocytochemical analysis of the aspiration cytologic specimens to the biochemical analysis of the excised tumor mass revealed a sensitivity and specificity of 80% and 89%, respectively. There was a significant semiquantitative correlation between the methods. These results extend the observation that antireceptor monoclonal antibody in immunohistochemical analysis is an effective tool in the evaluation of estrogen receptor content in human breast lesions and with careful specimen handling and proper controls may be extended to the evaluation of fine needle aspiration biopsies.

Antibodies, Monoclonal↗

PubMed1986

Ventricular shunt therapy of the brain: long-term rubber-catheter-induced inflammation.

Three cases of catheter-induced inflammation are presented, two of which involved brain parenchyma and the ventricular system and one that involved the soft tissues of the scalp. All of the catheters were composed of natural rubber and had been in place for several years. Abnormalities seen on computed tomographic scans in these patients were variable but included mass effect, vasogenic edema, catheter destruction, calcification, and abnormal contrast enhancement of the parenchyma adjacent to the catheters. Certain features on the scans of the two patients with intracranial disease, however, were common to both cases, allowing the correct preoperative diagnosis to be made in the second patient encountered.

Adult↗

PubMed1985

Two radioassays for serum vitamin B12 and folate determination compared in a reference interval study.

For 154 subjects, we verified that vitamin B12 and folate status was normal, using as criteria the average polymorphonuclear lobe count, mean corpuscular volume, and hemoglobin concentration. We then used blood from these subjects to compare values obtained with two radioassay kits, each designed for simultaneous vitamin B12 and folate determination. Although regression analysis showed reasonable correlation between the folate (r = 0.87) and vitamin B12 (r = 0.94) kits, we observed significant differences in the overall mean values for vitamin B12 (p less than 0.01) and folate (p less than 0.001) as measured with the kits in this population. Radioassay standard-curve data for the folate assays were similar, but these data indicated greater sensitivity at low concentrations for one vitamin B12 kit than the other. Using reference intervals recommended in the kit inserts, we found that the vitamin B12 status for 9% of these subjects would have been misclassified by one kit, 2% by the other.

Anemia, Megaloblastic↗

PubMed1984

Pseudocystic liver metastases in patients with carcinoid tumors: report of three cases.

Three of 30 patients (10%) with carcinoid tumors metastatic to the liver had large hepatic pseudocysts. One of the patients had an atypical carcinoid tumor that was markedly deficient in aromatic aminoacid decarboxylase and thus secreted 5-hydroxytryptophan as well as ACTH. Postmortem examination of this patient suggested that the mechanism of pseudocyst formation was probably infarction with liquefaction necrosis of tumor nodules. The authors' observations suggest that carcinoid tumors metastatic to the liver may not infrequently result in hepatic pseudocysts.

5-Hydroxytryptophan↗