Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability.

A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male "test" cells and 1 to 2 x 10(5) "compromised" female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression.

Sequencing radiation and adriamycin exposures in spheroids to maximize therapeutic gain.

Chinese hamster V79 spheroids were used as a tumor model to investigate sequence effects for combination treatments with adriamycin and radiation. Using cell sorting techniques, the subpopulation of cells most resistant to each modality as a single agent was identified, and combination treatments were then evaluated with the intent of identifying protocols leading to enhanced efficacy against the cell subpopulations that normally limited the success of single-agent treatments. Pre-irradiation drug exposure had the greatest therapeutic potential, not due to drug/radiation interactions, but rather, to drug-induced spheroid reoxygenation.

An enzyme-linked immunosorbent assay for erythropoietin using monoclonal antibodies, tetrameric immune complexes, and substrate amplification.

We recently reported the development of several monoclonal antibodies (MoAbs) to native human erythropoietin (Ep). In the present study we have used the two antibodies with highest affinity to develop a two-sided or sandwich enzyme-linked immunosorbent assay (ELISA) to measure Ep in human serum. In this assay Ep is incubated in microtiter wells precoated with the first (IgE) anti-Ep antibody.

Automated detection and recognition of live cells in tissue culture using image cytometry.

An automated image cytometry device, the Cell Analyzer, was used to locate live V79 cells plated at low densities in a tissue culture flask. Cells and other objects were detected by moving the flask in steps across a linear solid-state image sensor. The step size was selected to be small enough to allow detection of all the cells in the area being scanned but sufficiently large so that most cells would be detected on only one image line.

Inactivation of hypoxic cells by cisplatin and radiation at clinically relevant doses.

We have examined the effects of exposure to cisplatin (cis-diamminedichloroplatinum(II] on the response of exponentially growing V79 cells to low (0-4 Gy) and high (up to 30 Gy) doses of X rays under hypoxic and aerobic conditions. Survival in both dose regions was assessed by clonogenic assays; the low-dose studies were facilitated by a Cell Analyser (B. Palcic and B.

The interaction of trans-DDP [PtCl2(NH3)2] with low doses of radiation in mammalian cells.

Trans-DDP, a less toxic isomer of cisplatin, was examined for its radiosensitizing activity at high and low doses of ionizing radiation. Cells were exposed to the drug to produce low toxicity before exposure to X rays. A sensitive assay using the DMIPS Cell Analyzer was employed to measure cell survival response in low dose region (0-4 Gy) and conventional assay was used for high doses (4-25 Gy).

Potentiation of the tumor cytotoxicity of melphalan by vasodilating drugs.

Previous studies have shown that several vasoactive drugs can selectively reduce blood flow and increase hypoxia in experimental tumor systems. Our studies with one such agent, the vasodilator hydralazine, have clearly demonstrated that it can increase the tumor cytotoxicity of drugs which are known to be more toxic under hypoxic conditions. We have now extended our investigations to determine whether such selective reductions in tumor blood flow induced by hydralazine can increase the tumor cytotoxicity of other classes of cancer chemotherapeutic drugs.

Application of a silver-binding assay to the determination of protein in cerebrospinal fluid.

We evaluated a silver-binding assay for use in measuring total protein in cerebrospinal fluid. The advantage of this procedure over other methods is that, because of its sensitivity, it requires only a 0.5-microL sample.

The use of fluorescent probes to identify regions of transient perfusion in murine tumors.

Sequential intravenous injection of two fluorescent stains, Hoechst 33342 and DiOC7(3), can be used to quantify transient perfusion in experimental tumors. Regions of unmatched staining, indicative of intermittent perfusion, occur when vessels open or close in the 20 minute interval between administration of the dyes. In the murine SCCVII carcinoma, 8.

Interleukin-3 down-regulates its own receptor.

To gain insight into the mechanisms involved in regulating murine interleukin-3 (mIL-3) receptor expression, we have examined the effects of mIL-3 and murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) on mIL-3 receptor internalization and re-expression and studied the relationship between mIL-3 cell surface receptor density and growth factor sensitivity. As a source of cells for our studies, we used a B6SUtA clone, B6SUtA1, which grows equally well in mIL-3 or mGM-CSF when supplemented with 20% fetal calf serum (FCS) in RPMI 1640. Intracellular processing studies carried out in the presence and absence of methylamine suggested that mIL-3 is cleaved at two specific sites before its complete digestion within lysosomes.

Oxygen enhancement ratio of fractionated regimens in vitro.

The oxygen enhancement ratio (OER) of proliferating and nonproliferating cells grown in vitro was measured using accelerated fractionated regimens. Irradiations were performed either twice daily or three times per day, with a minimum of 6 h between the consecutive fractions. The dose delivered was 2.

Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48.

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown.

Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line.

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF.

Distribution and activity of antineoplastic drugs in a tumor model.

Antineoplastic drugs can be effective in solid tumors only if they can penetrate several cell layers and retain their activity in the tumor microenvironment. The capacity of several common chemotherapeutic agents to meet these requirements was evaluated in an in vitro tumor model, V79 Chinese hamster cells grown as spheroids. The delivery and toxicity of radioactively labeled 5-fluorouracil, lomustine, tetraplatin, and chlorambucil were determined by use of cell-sorting techniques to select cells as a function of their position (depth) within these spheroids, and the delivery and toxicity of doxorubicin (DOX) were evaluated on the basis of fluorescence intensity.

Reduction of tumour blood flow by vasoactive drugs: a role in cancer therapy.

The potential use of tumour blood flow reductions, induced by the vasodilator hydralazine, in cancer therapy are described. Data obtained in experimental tumour systems indicate that with appropriate scheduling and drug combinations, hydralazine can increase the therapeutic effectiveness of certain chemotherapeutic agents whether used alone or in combination with other modalities such as radiation or hyperthermia.

Evidence for intermittent radiobiological hypoxia in experimental tumour systems.

This paper describes flow and static fluorescence cytometry techniques to visualize and quantitate acute radiobiological hypoxia resulting from transient fluctuation in tumour blood flow in experimental tumour systems. The application of these techniques in two murine tumour systems provides evidence that such hypoxia exists and reduces the effectiveness of single doses of radiation. Possible mechanisms for and implications of these findings are discussed.

Tumor resistance to therapy: a genetic or kinetic problem?

Chinese hamster V79 spheroids exposed to cisplatin for 2 hr daily for 3 weeks responded very similarly to tumors undergoing chemotherapy. Initially, the number of viable cells per spheroid decreased in a dose-dependent fashion but, after several treatments, the apparent effectiveness of the cisplatin decreased. At low doses, spheroid regrowth eventually occurred despite continued therapy.

Autocrine production of pre-B-cell stimulating activity by a variety of transformed murine pre-B-cell lines.

Current evidence suggests that the proliferation and differentiation of normal pre-B-cells may be regulated by interactions with mesenchymally derived stromal cells. The nature of these cell-cell interactions has not yet been fully elucidated, but the involvement of pre-B-cell stimulating factors produced by various mesenchymal cell lines has recently been demonstrated in the murine system. In this model, transformed pre-B-cells differ from their normal counterparts in their acquisition of autonomous growth potential, as seen by an ability to be maintained in vitro in the absence of mesenchymal cell feeders.

Imaging system for morphometric assessment of absorption or fluorescence in stained cells.

An image acquisition and processing system has been developed for quantitative microscopy of absorption or fluorescence in stained cells. Three different light transducers are used in the system to exploit the best characteristics of these sensors for different biological measurements. A digital scanner, in the form of a linear array charge-coupled device (CCD), acquires data with high spatial and photometric resolution.

Repair and proliferation: major determinants of the multifraction radiation response.

An analysis of multifraction radiation survival data for V79 monolayers and spheroids, in which measured responses to each treatment were intercompared with the alpha/beta ratio derived using the common "reciprocal-dose" methodology at selected survival levels, resulted in quantitatively and qualitatively different conclusions. These discrepancies were the result of non-constant toxicity of each radiation dose in the multifraction scheme, which was obvious in the multifraction dose response curves, but not in the reciprocal dose plots. These data appear to indicate incomplete recovery between radiation exposures.

Measurement of cell motility and morphology with an automated microscope system.

A method is presented which provides quantitative descriptions of the morphology and motility behaviour of a large number of unperturbed individual cells. For that, a microscope system has been developed that performs automated tracking of motile mammalian cells in tissue culture and the concurrent measurement of cell morphology. Experiments using 3T3 mouse fibroblasts show that simple global morphological features, such as degree of cell elongation, correlate with the motile behaviour of individual cells.

Cytogenetic studies of haemopoietic colonies from patients with an initial diagnosis of acute lymphoblastic leukaemia.

For patients with an initial diagnosis of Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukaemia (ALL) and no documented history of Ph1-positive chronic myeloid leukaemia (CML), the cell of origin and extent of lineage involvement of the disease is often unclear. This is largely due to the fact that cytogenetic analysis of direct marrow preparations cannot distinguish between the presence of Ph1-positive myeloid metaphases and infiltration of the marrow with dividing Ph1-positive blasts. Cytogenetic analysis of cultured haemopoietic colonies allows more precise lineage assignment.

Postirradiation modification of tumor blood flow: a method to increase the effectiveness of chemical radiosensitizers.

The effect of postirradiation hypoxia induced by administration of the vasodilator hydralazine on the efficacy of misonidazole and RSU-1069 used in combination with radiation has been evaluated. Studies with the Lewis lung carcinoma indicate that hydralazine at a dose of 5 mg/kg reduces tumor blood flow and consequently increases the amount of hypoxia in the tumor tissue. Administration of hydralazine immediately after radiation treatment increased the amount of cell kill.

Regulation of hemopoietic progenitor cell proliferation.

Utilization of appropriate colony scoring criteria allows the identification of subsets of high proliferative potential, but lineage-restricted, erythropoietic and granulopoietic progenitors in human marrow. These cells share with human pluripotent hemopoietic cells the unique ability to vary their proliferative status from a non-cycling (GO) state to a cycling one (continuous progression from G1----S----G2----M). Time course studies of the size and turnover of these primitive, normally quiescent human hemopoietic cell populations in long-term marrow cultures has provided evidence that marrow mesenchymal elements play a role not only in supporting their maintenance, but also in regulating their proliferation.

Use of a sensitive bioimmunoabsorbent assay to isolate and characterize monoclonal antibodies to biologically active human erythropoietin.

At present, one of the most sensitive assays for human erythropoietin (Ep) is a bioassay that measures the Ep-dependent proliferation of spleen cells from phenylhydrazine-treated mice after 24 hours in culture. We describe how this assay can be used as the basis of a very sensitive method for detecting mouse antibodies to biologically active human Ep. In this procedure, microtiter wells are first coated with goat anti-mouse Ig antibody, then treated with mouse antibodies (serum or hybridoma culture supernatants), and finally incubated with a fixed amount of pure human Ep.