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Biomedical subjects

P Keng

Publications and source records attributed to P Keng.

27 records · Page 2Linked to original sources
PubMed1988

Separation of spontaneously differentiating and cell cycle-specific populations of HL-60 cells.

The human leukemia cell line HL-60 consists predominantly of abnormal promyelocytes. When grown in RPMI-1640 and 10% FCS between 5 and 10% of these cells spontaneously differentiate into more mature myeloid cells, becoming smaller in size and developing the ability to generate superoxide. Centrifugal elutriation was used to separate these G0 cells from the bulk of the cycling G1, S and G2M cells. These isolated differentiating cells are shown to be similar in size, DNA content, RNA content and NBT positivity not only to granulocyte induced HL-60 cells but also to human peripheral blood granulocytes. This methodology allows the study of differentiative vs proliferative processes through the quick one-step generation of homogeneous subpopulations of G0, G1, S and G2M cells.

Cell Cycle↗
PubMed1987

Macrophages are required for influenza virus infection of human lymphocytes.

Monocyte and lymphocyte surface-expressed viral antigens have been demonstrated after exposure of unseparated human mononuclear leukocytes to influenza virus in vitro. The current studies, using [35S]methionine pulse-labeled purified preparations of virus-exposed macrophages, depleted of lymphocytes, demonstrate that the presence of these viral proteins does represent new synthesis. However, purified lymphocytes, depleted of monocytes-macrophages and exposed to influenza virus, showed no detectable viral protein synthesis. In further experiments, unseparated mononuclear leukocytes were exposed to virus and subsequently separated by countercurrent centrifugal elutriation. Both macrophages and lymphocytes were then shown to synthesize influenza proteins. Cell-free control or influenza virus-infected macrophage-derived supernatant fluids did not facilitate influenza virus infection of the lymphocytes. The data suggest that macrophages are required for influenza virus infection of human lymphocytes, and raise the possibility that macrophage facilitation of an abortive infection of lymphocytes plays a role in the generation of effective immunity to viral antigens.

Antigens, Viral↗
PubMed1986

Lymphoma models for B cell activation and tolerance. III. Cell cycle dependence for negative signalling of WEHI-231 B lymphoma cells by anti-mu.

WEHI-231 B lymphoma cells have proven to be a useful model for the regulation of growth of normal B cells by anti-Ig reagents. We previously reported that the growth of these lymphoma cells is inhibited by heterologous or monoclonal anti-mu or anti-kappa reagents. Such cells cease to incorporate thymidine within 24-48 h of exposure to anti-Ig reagents, but are not adversely affected by antibodies directed at either class I or class II histocompatibility antigens. In fact, cell cycle analysis revealed that anti-mu causes a block in the transition of these cells from G1 to S phase. To further study the mechanism of growth inhibition, we have purified lymphoma cells in G1 by centrifugal elutriation, or enriched WEHI-231 cells at the G1/S interface by treatment with hydroxyurea, and followed their progression through the cell cycle in the presence or absence of anti-mu. Our data show that WEHI-231 B lymphoma cells receive a negative signal early in G1, since delayed addition of anti-mu (to late G1 cells) leads to no alteration in cell cycle progression at 24 h, and exposure to anti-mu during S does not alter progress through DNA synthesis and mitosis. Moreover, exposure to anti-mu for only 2 h prevents purified G1 cells from entering their first S phase. The nature of the relevant processes in early G1 is discussed in terms of models of B cell activation and tolerance induction.

Animals↗
PubMed1985

Lymphoma models for B-cell activation and tolerance. II. Growth inhibition by anti-mu of WEHI-231 and the selection and properties of resistant mutants.

Regulation of the growth of murine B-cell lymphomas has been used as a model for tolerance induction. The inhibition by anti-immunoglobulin reagents of the growth of WEHI-231 and several variant clones has now been studied. The parental line is exquisitely sensitive to growth inhibition by heterologous or monoclonal anti-mu or anti-k reagents and ceases to incorporate thymidine within 24-48 hr of exposure to anti-immunoglobulin reagents. Growth inhibition is initially reversible, but prolonged exposure to anti-mu results in cell death. This inhibition is specific for immunoglobulin light and heavy chains since growth is not inhibited by antibodies directed at either class I or class II histocompatibility antigens. In order to study the mechanism of growth inhibition, we have mutagenized WEHI-231 with ethylmethane sulfonate and cloned the surviving colonies in the presence of anti-mu. Such variants, which have been repeatedly recloned, are able to grow normally in the presence of anti-mu up to 100 micrograms/ml. These "resistant" clones, while expressing amounts of surface IgM similar to that observed on WEHI-231, do not differ markedly in their ability to cap their immunoglobulin receptors compared to the parental line but appear to have lost class II antigens. Cell cycle analysis revealed that anti-mu causes a block in the transition of WEHI-231 from G1 to S phase. The relevance of these processes to models of B-cell tolerance induction are discussed.

Animals↗
PubMed1985

The interaction between bleomycin and radiation on cell survival and DNA damage in mammalian cell cultures.

Chinese hamster ovary (CHO) cells were exposed to various concentrations of bleomycin (BLM) for 0.5, 1, 2, 4, 6, or 24 hours, followed with graded doses (0 to 800 rad) or radiation. The response to this chemotherapy-radiation combination treatment was measured by cell survival studies and DNA damage as determined by alkaline elution assay. Isobolograms for 1 and 2 log cell kills showed that cytotoxicity from BLM and from radiation were additive at 4, 15 and 75 milliunit/ml (mu/ml) for 1 or 4 hour exposure. When the exposure time of BLM was extended to 24 hours, slightly supraadditivity of lethality was found for the combination treatment with pharmacologically pertinent concentrations of 4 or 15 mu/ml. Therefore 4, 15 and 75 mu/ml concentrations of BLM all interacted positively with all doses of radiation to give enhanced cell kill. The alkaline elution patterns from the BLM-radiation combination also showed an enhancing effect on single strand breaks of DNA from treated cells. A human oat cell carcinoma cell line (MEMAR cells) was also studied and found to be more sensitive to BLM than were CHO cells.

Animals↗
PubMedDate not supplied

In vitro hypoxic cytotoxicity of nitroimidazoles: uptake and cell cycle phase specificity.

The hypoxic cytotoxicity of four different 2-nitroimidazoles of similar electron affinities but different lipophilicities was compared using EMT6/Ro mouse mammary tumor cells in exponential growth phase in severely (less than 20 ppm) hypoxic conditions. The relative cytotoxicities were misonidazole (MISO) = desmethylmisonidazole (9963) greater than SR-2508 much greater than SR-2555 indicating that the compounds with the lowest lipophilicity were less cytotoxic. The rates of uptake of these compounds were MISO greater than 9963 greater than SR-2508 = SR-2555. These data together with comparisons of the amounts of cell-associated compounds indicate that the similarity in toxicity of MISO and 9963 can be related to a general similarity in their pharmacokinetics, but that other unknown factors must be considered to explain the relative toxicity of SR-2508 and SR-2555. In other experiments, EMT6/Ro cells synchronized using centrifugal elutriation were most sensitive in hypoxia to MISO at the late G1--early S phase of the cell cycle. These data indicate the importance of considering cellular and subcellular distribution of these nitroimidazoles as well as possible cell cycle specificity for cytotoxicity in interpreting relative effectiveness of different compounds in responses of mixed populations of cells in cultures or tumors.

Animals↗
PubMedDate not supplied

Engineering a human bone marrow model: a case study on ex vivo erythropoiesis.

Bone marrow, with its intricate, three-dimensional tissue structure facilitating cell-cell interactions, provides a microenvironment supporting the production of hundreds of billions of multilineal blood cells everyday. We have developed a three-dimensional bone marrow culture system in which marrow cells are cultured in a reactor packed with porous microspheres. The culture supports a three-dimensional growth configuration and multilineal hemopoiesis mimicking the bone marrow in vivo. We studied ex vivo human erythropoiesis using the three-dimensional culture system. The system sustained extensive erythropoiesis at low erythropoietin concentrations (0.2 U/mL), plus stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and insulin-like growth factor-I. Erythroid cell production lasted for more than 5 weeks, and the percentage of erythroid cells in the nonadherent cell population was approximately 60%. Flow cytometric analysis using cell surface markers specific for erythroid cells (CD71 and glycophorin-A) indicated that the culture produced early, intermediate, and late erythroid cells. As the culture progressed, the erythroid cell population shifted gradually toward mature cell types. When compared to the three-dimensional culture, the traditional flask cultures failed to support extensive erythropoiesis under the same conditions. This indicates that the three-dimensional bone marrow culture system provides a microenvironment conducive to erythropoiesis under more physiological conditions and is a better bone marrow model.

Biomedical Engineering↗
PubMedDate not supplied

Oncogene elements within an endogenous retrovirus.

The human genome contains a large number of endogenous retroviral-related sequences. While the function of these sequences is unknown, they may contribute to disease processes through their regions of homology with infectious retroviruses. We have been further characterizing a recently reported HTLV-1 related endogenous retroviral sequence cloned from T lymphocytes isolated from a patient with essential cryoglobulinemia. We here report further detailed transcriptional analysis of the sequence for tissue and cell-cycle specificity and a novel finding of an association between the endogenous retrovirus and a ras-related gene.

Base Sequence↗