Robert E. Gallagher

Bio: Robert E. Gallagher is an academic researcher from National Institutes of Health. The author has contributed to research in topics: Virus & Cell culture. The author has an hindex of 11, co-authored 16 publications receiving 4890 citations. Previous affiliations of Robert E. Gallagher include University of Maryland Marlene and Stewart Greenebaum Cancer Center.

Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture.

Abstract: ATTEMPTS to develop long-term suspension cultures of human myeloid leukaemic cells have met with limited success. Lymphoblastoid lines carrying the Epstein–Barr virus genome occasionally arise during such attempts but these lymphoid cells originate from contaminating B lymphocytes and not from the leukaemic myeloid cells1. A line established from the pleural fluid of a patient with chronic myeloid leukaemia in blast crisis2 (designated K-562) has no B-cell or T-cell markers3–4 and does not seem to be of lymphoid origin4. Its lack of morphological and histochemical differentiation2–4, however, makes it difficult to determine whether these cells are derived from myeloblasts or more primitive stem cells4. Another less documented cell line (8261) derived from the peripheral blood of a patient with acute myelogenous leukaemia showed apparent morphological and functional differentiation in agar in the presence of a feeder layer of peripheral blood leukocytes but did not differentiate in suspension culture5. Our laboratory previously reported that cultures of differentiating myeloid leukaemic cells can be maintained for several months in suspension culture but only when enriched with conditioned media (CM) from certain monolayer fibroblastic cultures of first trimester whole human embryos (ref. 6 and Ruscetti et al. in preparation). We describe here for the first time the derivation from myeloid leukaemic cells of a leukocyte culture that by morphological and histochemical criteria clearly and persistently differentiates along the myeloid series without an exogenous source of conditioned medium.

Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.

Abstract: A human leukemic cell line (designated HL-60) has recently been established from the peripheral blood leukocytes of a patient with acute promyelocytic leukemia. This cell line displays distinct morphological and histochemical commitment towards myeloid differentiation. The cultured cells are predominantly promyelocytes, but the addition of dimethyl sulfoxide to the culture induces them to differentiate into myelocytes, metamyelocytes, and banded and segmented neutrophils. All 150 clones developed from the HL-60 culture show similar morphological differentiation in the presence of dimethyl sulfoxide. Unlike the morphologically immature promyelocytes, the dimethyl sulfoxide-induced mature cells exhibit functional maturity as exemplified by phagocytic activity. A number of other compounds previously shown to induce erythroid differentiation of mouse erythroleukemia (Friend) cells can induce analogous maturation of the myeloid HL-60 cells. The marked similarity in behavior of HL-60 cells and Friend cells in the presence of these inducing agents suggests that similar molecular mechanisms are involved in the induction of differentiation of these human myeloid and murine erythroid leukemic cells.

Infective transmission and characterisation of a C-type virus released by cultured human myeloid leukaemia cells

Abstract: A C-type virus isolated from long term cultures of myeloid cells from a patient with acute myelogenous leukaemia is infectious for a wide variety of cells. The establishment of chronically infected cells enabled us to characterise the virus by biological, immunological, and biochemical tests. The virus is closely related to the simian sarcoma-associated virus isolated from a woolly monkey fibrosarcoma.

Growth and differentiation in culture of leukemic leukocytes from a patient with acute myelogenous leukemia and re-identification of type-C virus.

Abstract: Conditioned medium from a culture of whole human embryo cells stimulated prolonged exponential growth in suspension culture of leukocytes from a patient with acute myelogenous leukemia. Ten to 20% of the cultured cells consistently differentiated into mature granulocytes including neutrophils, eosinophils, and basophils. The proportion of lymphocytes declined after culturing, and tests for Epstein-Barr virus antigens were negative. An abnormality of a G group chromosome was observed in some metaphases from the patient's fresh bone marrow and from the cultured leukocytes, indicating growth in vitro of leukemic cells. After 4-10 weeks in culture, a budding type-C virus was continuously released by the cultured leukocytes, predominantly by undifferentiated blast cells. This virus was originally identified in three different cultures of a peripheral blood specimen obtained at the time of diagnosis. Subsequently, this virus was identified by reverse transcriptase (RNA-dependent DNA polymerase) assays and by electron microscopy in cultured leukocytes from a bone marrow specimen obtained 14 months later from the same patient. Virus produced by cultures of both specimens was closely related, if not identical, to the woolly monkey type-C virus.

Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma

Abstract: Retrovirus particles with type C morphology were found in two T-cell lymphoblastoid cell lines, HUT 102 and CTCL-3, and in fresh peripheral blood lymphocytes obtained from a patient with a cutaneous T-cell lymphoma (mycosis fungoides). The cell lines continuously produce these viruses, which are collectively referred to as HTLV, strain CR(HTLVCR). Originally, the production of virus from HUT 102 cells required induction with 5-iodo-2′-deoxyuridine, but the cell line became a constitutive producer of virus at its 56th passage. Cell line CTCL-3 has been a constitutive producer of virus from its second passage in culture. Both mature and immature extracellular virus particles were seen in thin-section electron micrographs of fixed, pelleted cellular material; on occasion, typical type C budding virus particles were seen. No form of intracellular virus particle has been seen. Mature particles were 100-110 nm in diameter, consisted of an electron-dense core surrounded by an outer membrane separated by an electron-lucent region, banded at a density of 1.16 g/ml on a continuous 25-65% sucrose gradient, and contained 70S RNA and a DNA polymerase activity typical of viral reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase). Under certain conditions of assay, HTLVCR RT showed cation preference for Mg2+ over Mn2+, distinct from the characteristics of cellular DNA polymerases purified from human lymphocytes and the RT from most type C viruses. Antibodies to cellular DNA polymerase γ and anti-bodies against RT purified from several animal retroviruses failed to detectably interact with HTLVCR RT under conditions that were positive for the respective homologous DNA polymerase, demonstrating a lack of close relationship of HTLVCR RT to cellular DNA polymerases γ or RT of these viruses. Six major proteins, with sizes of approximately 10,000, 13,000, 19,000, 24,000, 42,000, and 52,000 daltons, were apparent when doubly banded, disrupted HTLVCR particles were chromatographed on a NaDodSO4/polyacrylamide gel. The number of these particle-associated proteins is consistent with the expected proteins of a retrovirus, but the sizes of some are distinct from those of most known retroviruses of the primate subgroups.

Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay

Abstract: For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

Identification of the cystic fibrosis gene: Chromosome walking and jumping

Abstract: An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.

Establishment and characterization of a human acute monocytic leukemia cell line (THP‐1)

Abstract: A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia is described. This cell line had Fc and C3b receptors, but no surface or cytoplasmic immunoglobulins. HLA haplotypes of THP-1 were HLA-A2, -A9, -B5, -DRW1 and -DRW2. The monocytic nature of the cell line was characterized by: (1) the presence of alpha-naphthyl butyrate esterase activities which could be inhibited by NaF; (2) lysozyme production; (3) the phagocytosis of latex particles and sensitized sheep erythrocytes; and (4) the ability to restore T-lymphocyte response to Con A. The cells did not possess Epstein-Barr virus-associated nuclear antigen. These results indicate that THP-1 is a leukemia cell line with distinct monocytic markers. During culture, THP-1 maintained these monocytic characteristics for over 14 months.

Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture.

Abstract: ATTEMPTS to develop long-term suspension cultures of human myeloid leukaemic cells have met with limited success. Lymphoblastoid lines carrying the Epstein–Barr virus genome occasionally arise during such attempts but these lymphoid cells originate from contaminating B lymphocytes and not from the leukaemic myeloid cells1. A line established from the pleural fluid of a patient with chronic myeloid leukaemia in blast crisis2 (designated K-562) has no B-cell or T-cell markers3–4 and does not seem to be of lymphoid origin4. Its lack of morphological and histochemical differentiation2–4, however, makes it difficult to determine whether these cells are derived from myeloblasts or more primitive stem cells4. Another less documented cell line (8261) derived from the peripheral blood of a patient with acute myelogenous leukaemia showed apparent morphological and functional differentiation in agar in the presence of a feeder layer of peripheral blood leukocytes but did not differentiate in suspension culture5. Our laboratory previously reported that cultures of differentiating myeloid leukaemic cells can be maintained for several months in suspension culture but only when enriched with conditioned media (CM) from certain monolayer fibroblastic cultures of first trimester whole human embryos (ref. 6 and Ruscetti et al. in preparation). We describe here for the first time the derivation from myeloid leukaemic cells of a leukocyte culture that by morphological and histochemical criteria clearly and persistently differentiates along the myeloid series without an exogenous source of conditioned medium.