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Biomedical subjects

A Keating

Publications and source records attributed to A Keating.

At least 145 records · Page 8Linked to original sources
PubMed1989

Synergistic effect of ketoconazole and antineoplastic agents on hormone-independent prostatic cancer cells.

Ketoconazole has been recently used in the primary treatment of patients with metastatic cancer of the prostate and is identified as a potent inhibitor of cytochrome P450-dependent adrenal and testicular androgen production. The drug has also shown activity in patients failing conventional hormonal manipulation. We subsequently showed that ketoconazole in vitro has a direct cytotoxic effect on human androgen-independent prostatic cancer cell lines. In order to better define the possible role of ketoconazole on hormone-independent prostatic cancer, we incubated the cells from human androgen-independent prostatic cancer lines in a methylcellulose tumour colony assay with different doses of the drug and increasing doses of conventional cytotoxic agents (etoposide, bleomycin, vinblastine, methotrexate, and teniposide). We demonstrated synergistic suppression of prostate cancer clonogenic cell growth by ketoconazole in the presence of vinblastine or etoposide. This observation may assign a new and important role for ketoconazole as part of combination chemotherapy in the treatment of patients with advanced prostatic cancer.

Antineoplastic Agents↗
PubMed1988

Electric field-mediated gene transfer: characterization of DNA transfer and patterns of integration in lymphoid cells.

Southern analysis of individual transfectants generated by electroporation demonstrated a strong preference for the integration of DNA in low copy number even when electroporation was performed in the presence of increasing DNA concentrations. Although transfer of multiple DNA copies was detected at higher DNA concentrations (16 pmoles/ml or greater), the average gene copy number even at 36 pmoles DNA per ml, was only 13. Multiple gene copies were integrated at either a few chromosomal sites, or at a single site within individual transfectants. Restriction endonuclease cleavage data were consistent with a random orientation of molecules within a concatemer, suggesting that the concatemer may have risen via end-to-end ligation of linear molecules, rather than by homologous recombination. Integration of exogenous DNA into the host chromosome occurred preferentially at the ends of the linear molecule. Although the linearization site was lost upon integration, endonuclease sites as close as 18 bp from the linearization site were retained. These data, as well as direct restriction mapping of the transferred genes, indicate that DNA transfer and integration occur without DNA rearrangement. Taken together, these results suggest that electroporation may offer some unique advantages for the transfer of eukaryotic genes.

Animals↗
PubMed1988

Anticonvulsant-induced marrow suppression and immune thrombocytopenia.

Phenytoin and carbamazepine are rarely associated with serious hematologic side effects but can include impairment of either humoral or cell-mediated immunity. We describe a patient who developed severe granulocytopenia while taking phenytoin. The phenytoin was replaced by carbamazepine and the patient subsequently developed erythroid hypoplasia, neutropenia and persistent thrombocytopenia. In vitro studies demonstrated a phenytoin-dependent antigranulocyte antibody directly implicating phenytoin in the leukopenia. An extremely high titre of platelet-associated IgG was found which was independent of the presence of carbamazepine. Autoantibodies directed against the patient's red cells, granulocytes and lymphocytes were also demonstrated. In vitro marrow culture studies failed to detect cellular or humoral inhibitors and were suggestive of a stem cell defect. These studies indicate that anticonvulsant therapy can result in sustained humoral abnormalities as well as in nonimmune mediated marrow suppression.

Antigen-Antibody Complex↗
PubMed1988

Hierarchical organization of hematopoietic microenvironments: role of proteoglycans.

The orderly process of hematopoietic cell differentiation depends on the organization of specific microenvironments which function by assembling progenitor cells and appropriate concentrations of necessary growth factors. We propose that this organization involves a spectrum of microenvironmental complexity concomitant with changing cellular requirements, that it is achieved by the extracellular matrix produced by marrow stromal cells, and that its specificity is conferred by the heterogeneity intrinsic in the conformation of proteoglycans and their constituent glycosaminoglycans.

Animals↗
PubMed1987

Acute leukemia occurring 19 years after treatment of acute lymphoblastic leukemia.

A 22-year-old woman presented with acute lymphoblastic leukemia (ALL) during pregnancy. She had been successfully treated for ALL at the age of 3 years and had received maintenance treatment for 11 years. Complete remission of 3 years or more is an important factor for long-term survival and potential cure. Relapses may occur in the first 5 years after maintenance is discontinued. Later relapses are distinctly unusual. We have found six more cases of ALL occurring long after the initial remission in the English literature. It is unclear whether late relapse represents true relapse or a second leukemia. Life-long follow-up of children with ALL may be necessary.

Acute Disease↗
PubMed1987

Correlation between cytogenetic and molecular findings in human chronic myelogenous leukemia lines EM-2 and EM-3.

Few established cell lines derived from patients with chronic myelogenous leukemia have been reported. Cytogenetic examinations of two independently derived Philadelphia chromosome (Ph)-positive cell lines from a patient with chronic myelogenous leukemia were performed serially from their initiation in 1982 to the present. Subcultures of each of these lines maintained separately in two laboratories for over 2 years were compared for degree of divergence. The modal chromosome number declined substantially within the first few months and slowly thereafter. Despite hyperploidy, these lines have remained remarkably stable cytogenetically. For each line, the modal chromosome number is hypotetraploid, multiple copies of Ph are present and no normal chromosome #9 remains. Only a few marker chromosomes have arisen. Despite the multiple copies of Ph, a single bcr restriction pattern was seen, suggesting duplication of a single Ph, rather than independent translocation events. These lines should be very useful for in vitro studies of chronic myelogenous leukemia.

Cell Line↗
PubMed1987

Treatment of acute myeloid leukemia cells in vitro with a monoclonal antibody recognizing a myeloid differentiation antigen allows normal progenitor cells to be expressed.

Monoclonal antibody L4F3 reacts with most acute myeloid leukemia (AML) cells and virtually all normal granulocyte/monocyte colony-forming cells (CFU-GM). Our objective was to determine whether lysis of AML cells with L4F3 and complement allowed expression of normal myeloid progenitors. The five glucose-6-phosphate dehydrogenase (G6PD) heterozygous patients with AML studied manifested only a single G6PD type in blast cells and in most or all granulocyte colony-forming cells, indicating that the leukemias developed clonally. The cells remaining after L4F3 treatment from two of the patients gave rise to granulocytic colonies that expressed the G6PD type not seen in the leukemic clone, indicating that they were derived from normal progenitors (CFU-GM). L4F3-treated cells from these two patients cultured over an irradiated adherent cell layer from normal long-term marrow cultures also gave rise to CFU-GM, which were shown by G6PD analysis to be predominantly nonleukemic. In the other three patients, the progenitor cells remaining after L4F3 treatment were derived mainly from the leukemic clone. The data suggest that in vitro cytolytic treatment with L4F3 of cells from certain patients with AML can enable normal, presumably highly immature progenitors to be expressed.

Adolescent↗
PubMed1987

Simian virus 40-transformed adherent cells from human long-term marrow cultures: cloned cell lines produce cells with stromal and hematopoietic characteristics.

Adherent cells from long-term marrow cultures from 23 individuals were transformed with wild-type simian virus 40 (SV40). After transformation, cloned cell lines were developed that even after rigorous subcloning invariably produced both stromal cells and round cells. The stromal cells expressed cytoskeletal filaments similar to those of long-term marrow culture adherent cells and produced interstitial and basal lamina collagen types. The round cells had the electron microscopic appearance of primitive hematopoietic cells and when examined with cytochemical stains and monoclonal antibodies to hematopoietic differentiation antigens had reaction patterns suggestive of cells from several lineages. Most round cells expressed the pan-hematopoietic T-200 determinant, and lesser percentages expressed the early T cell antigens CD-1 and CD-3, HLA-DR determinants, the monocytic antigen recognized by Leu M3, and the myeloid antigens detected by monoclonal antibodies 1G10 and 12.8. In addition, when plated in semisolid medium in the presence of a source of colony-stimulating activity, up to 11% of the cells formed colonies consisting of blastlike cells that also expressed hematopoietic cell surface determinants. The data suggest that adherent cells in long-term marrow cultures contain a cell that after transformation by SV40 obligately produces cells with hematopoietic as well as stromalike features.

Bone Marrow↗
PubMed1986

The c-abl, bcr and C lambda genes are amplified in a cell line but not in the uncultured cells from a patient with chronic myelogenous leukemia.

Structural alterations of the oncogenes in human tumors are reported to result from a variety of mechanisms: point mutations, chromosomal translocations and gene amplifications. In over 90% of the cases of chronic myelogenous leukemia (CML), the c-abl oncogene is translocated from chromosome 9 to chromosome 22, and forms in part the Philadelphia (Ph1) chromosome. We have molecularly analysed a double Ph1-positive (Ph1+) cell line, KBM-5 that was established from a patient with CML in the blast-transformed phase (CML-BP). We report that the c-abl, bcr, and C lambda genes are amplified approximately eight-fold in the cell line but not in the fresh uncultured cells from which KBM-5 was derived.

Cell Line↗
PubMed1986

Stable expression of selectable genes introduced into human hematopoietic stem cells by electric field-mediated DNA transfer.

The successful introduction of DNA into human bone marrow cells by electric field-mediated transfer was initially demonstrated by the detection of transient chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2,3.1.28) activity in marrow cell extracts. To determine whether DNA was transferred into hematopoietic stem cells, human nucleated marrow cells were subjected to electroporation in the presence of a plasmid construct containing the bacterial genes conferring resistance to the neomycin analogue G418 (neo) and to mycophenolic acid (gpt). The growth of granulocyte/macrophage colonies in selective media, followed by hybridization analyses of resistant cells, established that DNA was transferred into human granulopoietic progenitor cells and was stably maintained and expressed in their differentiated progeny. Electroporation, therefore, offers the opportunity to transfer genes effectively into human hematopoietic stem cells and avoids some of the disadvantages associated with other methods of gene transfer.

Bone Marrow↗
PubMed1986

Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.

The technique of DNA transfer by electroporation was investigated in an effort to evaluate its utility for the identification of developmentally controlled regulatory sequences. Transient and stable gene expression was detected in a variety of lymphoid cell lines subjected to electroporation. No correlation existed between the levels of chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) expression and stable transfection frequency. In all lymphoid cell lines tested, the simian virus 40 early region was a better promoter than was the Rous sarcoma virus long terminal repeat.

Acetyltransferases↗
PubMed1986

Immunoreactive parathyroid hormone and in vitro inhibition of erythropoiesis by uremic serum in patients on continuous ambulatory peritoneal dialysis.

Inhibition of erythropoiesis in vitro by uremic serum is well established. The role of parathyroid hormone (PTH) as inhibitor of erythropoiesis is uncertain. To further elucidate this effect we correlated the in vitro growth of red cell progenitors (BFU-E) in the presence of uremic serum from 26 patients on continuous ambulatory peritoneal dialysis with levels of immunoreactive PTH in the serum. The patients had a mean hemoglobin level of 100 +/- 20.1 g/l. The BFU-E growth was 98.8 +/- 28.3% of control. No correlation between BFU-E growth and hemoglobin was found. A significant negative correlation between levels of immunoreactive PTH and BFU-E growth was found (p = 0.05). These results support the notion that PTH or PTH fragment(s) might be responsible for the in vitro inhibition of erythropoiesis by uremic serum in patients on continuous ambulatory peritoneal dialysis.

Erythropoiesis↗
PubMed1986

Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses.

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H-activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Bone Marrow↗
PubMed1985

Responsiveness of the in vitro hematopoietic microenvironment to platelet-derived growth factor.

The adherent or stromal cells in human long-term marrow cultures, a possible in vitro counterpart of the in vivo microenvironment, were investigated for responsiveness to platelet-derived growth factor (PDGF). Many stromal cells from cultures derived from normal donors as well as from patients with chronic myelogenous leukemia, bore receptors to PDGF and were stimulated to incorporate [3H]-thymidine by highly purified PDGF and to a lesser extent by epidermal growth factor (EGF). These data suggest that PDGF and perhaps EGF may be involved in the regulation of the in vitro microenvironment and, therefore, of normal and possibly neoplastic hematopoiesis.

Autoradiography↗
PubMed1985

A functional defect in irradiated adherent layers from chronic myelogenous leukemia long-term marrow cultures.

The adherent cell layer in the human long-term marrow culture system provides an in vitro model for the hematopoietic microenvironment. We have devised a two-stage long-term marrow culture system to examine the effect of adherent cell layers from normal and neoplastic cultures on early hematopoietic progenitors. We found that irradiated layers from normal long-term marrow cultures supported granulopoiesis in normal allogeneic marrow cells. This effect, manifested by increased CFU-C production, was amplified when the irradiated layers were overlaid with normal allogeneic marrow initially depleted of these stem cells with cytotoxic anti-Ia (HLA-DR) antibodies. In contrast, there was no regeneration of CFU-C from normal marrow depleted of these progenitors after incubation with irradiated layers from chronic myelogenous leukemia cultures or with passaged marrow fibroblasts. Our data suggest that a functional abnormality may exist in the in vitro microenvironment in chronic myelogenous leukemia and that the two-stage long-term marrow culture system may provide a means of assessing the capacity of adherent cells to support hematopoiesis and an indirect assay for stem cells not measurable with semisolid clonal methods.

Antibodies, Monoclonal↗
PubMed1985

CGA-7 and HHF, two monoclonal antibodies that recognize muscle actin and react with adherent cells in human long-term bone marrow cultures.

The CGA-7, a monoclonal antibody that reacts with smooth muscle cell actin but not with endothelial cell or fibroblast actin, and HHF, a monoclonal antibody that reacts with smooth muscle, skeletal muscle, and cardiac muscle actin, both recognize microfilaments present within adherent cells from actively hematopoietic human long-term marrow cultures. Macrophages, monocytes, and cultured marrow fibroblasts do not react with either antibody. These data suggest that the anti-actin antibodies may serve as useful markers for in vitro microenvironmental cells and lend support to the hypothesis that stromal cells from long-term marrow cultures are different from marrow fibroblasts and may constitute a unique cell lineage.

Actins↗