The response of hypoxic cells in SCCVII murine tumors to treatment with cisplatin and x rays.

Possible mechanisms of enhancement of radiation effects by cisplatin, including radiosensitization of hypoxic cells, drug-induced tumor reoxygenation, and inhibition of repair of sublethal radiation damage, were examined in the murine SCCVII model. Combination radiation/drug treatments were most effective when drug exposure preceded irradiation of animals breathing a reduced oxygen atmosphere, indicating that the primary interaction between the modalities was a cisplatin-induced increase in the oxygenation status of the acutely hypoxic cells in those tumors. Delivering cisplatin prior to or immediately after the first of two 5 Gy fractions was more effective than combinations with a single x-ray exposure, suggesting that proper sequences of the combined modalities may augment natural reoxygenation processes.

Keynote address: the influence of microenvironmental factors on the activity of radiation and drugs.

The inherent radio- and chemosensitivity of tumor cells clearly affects their response to treatment. Accumulating evidence, however, suggests that the biochemical and physiological status of the cell during treatment is at least as important. In this review, a critique of the current evidence for, and extent of, microenvironmental heterogeneity in tumors is presented, emphasizing human tumor cells in situ.

Interaction of platinum drugs with clinically relevant x-ray doses in mammalian cells: a comparison of cisplatin, carboplatin, iproplatin, and tetraplatin.

Whereas the interaction between radiation and platinum complexes has never been pronounced in radiobiological experiments (to 30 Gy in mammalian cells), there have been reports of interest in this combination in the clinic, where fractionated doses of approximately 2 Gy are used. Our studies on the marked interaction in hypoxia at the 80% survival level (1-2.5 Gy) with cisplatin have been extended to second generation platinum drugs of clinical interest.

Nicotinamide, Fluosol DA and Carbogen: a strategy to reoxygenate acutely and chronically hypoxic cells in vivo.

The effect of Nicotinamide and/or treatment with Fluosol DA and Carbogen breathing on the radiation response of 500-750 mg SCCVII and KHT tumours has been evaluated. Pretreatment with Fluosol DA/Carbogen or Nicotinamide resulted in relatively modest enhancements of radiation damage with enhancement factors of 1.1 and 1.

Drug induced perturbations in tumor blood flow: therapeutic potential and possible limitations.

Chemical modulation of tumor blood flow has until recently received relatively little attention as a therapeutic tool. Developments in the last few years, both in technology and in drug development, have changed this perspective. Fluorescence activated cell sorting techniques have provided evidence for the existence of acutely hypoxic cells resulting from transient fluctuations in microregional tumor blood flow in experimental tumor systems.

Physical basis for detection of DNA double-strand breaks using neutral filter elution.

Results using neutral filter elution are difficult to explain if this method detects only DNA double-strand breaks (DSBs). In an attempt to understand neutral filter elution, the size of DNA pieces eluted from filters was measured using pulsed-field gel electrophoresis. Contrary to expectation, the size of the pieces was independent of radiation dose and time of elution, and much smaller (approximately 460 kb) than anticipated based on the expected number of DSBs induced.

Explanted Fish Neural Tubes Give Rise to Differentiating Neural Crest Cells: (neural crest cell culture/pigment cells/Xiphophorus/medaka/fish).

Neural tubes were explanted from the trunk of various embryonic stages of three teleost fish, Xiphophorus maculatus (platyfish), X. helleri (swordtail), and Oryzias latipes (Japanese medaka) with the aim to obtain in vitro differentiating neural crest cells. Outgrowth of cells was observed immediately after attachment of the explants on dishes coated with fibronectin.

Modification of tumour radiation response in vivo by the benzamide analogue pyrazinamide.

Pyrazinamide, the pyrazine analogue of nicotinamide, has been evaluated for its ability to modify the radiation response of hypoxic cells both in vivo and in vitro. Results obtained with three different murine tumour systems EMT6, LLC and SCCVII showed that pyrazinamide at a dose of 0.5 mg g-1 i.

Expression cloning of a cDNA encoding M1/69-J11d heat-stable antigens.

The differentiation Ag identified by the mAb M1/69 and J11d (commonly referred to as heat-stable Ag) are found in structurally heterogeneous forms on the surfaces of many types of murine hemopoietic cells. The extinction of expression of these antigens is associated with thymocyte maturation and Ig class switching in B cells, as well as terminal differentiation of macrophages. A cDNA encoding the M1/69-J11d peptide was cloned from a hemopoietic progenitor cell line by immunoselection of COS cells transfected with expression libraries.

Detection and isolation of the erythropoietin receptor using biotinylated erythropoietin.

Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin.

Intermittent blood flow in the KHT sarcoma--flow cytometry studies using Hoechst 33342.

The administration of the fluorescent DNA stain, Hoechst 33342, to mice bearing the KHT sarcoma, combined with flow cytometry, can be used to select cells according to their proximity to functional vasculature. Different protocols of administration of Hoechst 33342 were used in order to differentiate between the presence of temporary and chronically hypoxic cells. The results show a large difference in radiosensitivity between cells close to, and distant from, functional vasculature.

Autonomous expression of RTVL-H endogenous retroviruslike elements in human cells.

Northern (RNA) blot analysis of RNA from various human cell lines and tissues has demonstrated that elements belonging to the RTVL-H family of human endogenous retroviruslike sequences are expressed in several cell types. The highest levels of RTVL-H-related RNAs were observed in teratocarcinoma cell line NTera2D1, HeLa cells, two bladder carcinoma cell lines, and normal amniotic tissue. Expression was also observed in normal chorion and in some other cell lines.

Multicell spheroids as a model for cell kinetic studies.

Cells growing in tissue culture as three-dimensional, multicellular aggregates called 'spheroids' typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour-like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described.

Radiosensitization by metal complexes of 4(5)-nitroimidazole.

Four closely-related cis-platinum (Pt) complexes of 4(5)-nitroimidazole have been examined with respect to properties of radiobiological interest, to test the hypothesis that targeting a nitroimidazole (NO2Im) to DNA could enhance its radiosensitizing ability: I [PtCl2(5-NO2Im)2]; II [PtCl2(4-NO2Im)2]; III [PtCl2(NH3)(5-NO2Im)]; IV [PtCl2(NH3)(4-NO2Im)]. The reduction potential was affected to the same extent on metal binding in all of the complexes (delta E1/2 = +200 mV, cf. ligand measured polographically).

Slow penetration of anthracyclines into spheroids and tumors: a therapeutic advantage?

Despite clear evidence that the effective penetration of the anthracycline antibiotics into experimental tumors or multicell spheroids is poor, these drugs exhibit clinical activity against a variety of solid tumors. In an attempt to understand this apparent contradiction, we used the Chinese hamster V79 spheroid system and flow cytometry techniques for intra-spheroid pharmacological studies of doxorubicin and daunomycin. Our results indicate that the slow delivery of the anthracyclines to the inner cells of spheroids is due to the rapid binding of the drug by cells in the outer layers.

Preclinical evaluation of pions in vivo: experience at TRIUMF.

This paper describes the results obtained from in vivo studies of the pion beam at the TRI University Meson Facility (TRIUMF). The studies encompass work (from 1978 to 1986), designed to evaluate the RBE for early and late effects and to assess the importance of X-ray dose rate and treatment volume on these values. Results with early responding tissues, i.

Effect of focus on cell detection and recognition by the Cell Analyzer.

The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 microns below to 25 microns above the optimal focus position.

Synergism of cisplatin and mitomycin C in sensitive and resistant cell subpopulations of a tumor model.

Cisplatin is a widely-used anti-neoplastic agent with activity against a broad spectrum of human solid tumors. It is, however, seldom curative as a single agent. In an in vitro tumor model system, V79 spheroids, the non-cycling, hypoxic cell subpopulations are most resistant to cisplatin, suggesting that combination chemotherapy with cisplatin and a drug which preferentially killed hypoxic cells might prove useful.

High-efficiency gene transfer to human hematopoietic cells maintained in long-term marrow culture.

We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony-stimulating factor and interleukin-1 beta or medium conditioned by a human marrow "stromal" cell line to medium conditioned by agar-stimulated human leukocytes both during the infection and the colony growth period.

Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.

To develop a purification strategy for isolating the most primitive hematopoietic stem cells present in normal human marrow we have combined cell separation techniques with an assay for cells that initiate sustained hematopoiesis in vitro in the presence of irradiated human marrow adherent cells. These "feeders" were established by subculturing 2- to 6-week-old primary long-term marrow culture adherent layers at a density of 3 x 10(4) irradiated cells per square centimeter. Test "long-term culture (LTC)-initiating cells" were plated on top of the feeders and the cocultures then maintained as standard long-term marrow cultures with half-media changes and removal of half of the nonadherent cells each week.

Histological evidence for nonperfused vasculature in a murine tumor following hydralazine administration.

The effect of the vasodilator hydralazine on tumor vascular function has been evaluated in C3H/He mice bearing subcutaneously implanted SCCVII squamous cell carcinoma. Changes in microregional perfusion following hydralazine administration were observed using a double fluorescent staining technique. Hydralazine-induced alterations in tumor blood flow were measured using laser Doppler flowmetry.

Identification and characterization of 114/A10, an antigen highly expressed on the surface of murine myeloid and erythroid progenitor cells and IL-3-dependent cell lines.

Monoclonal antibody (mAb) 114/A10, raised against the murine bone marrow-derived multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by various interleukin-3 (IL-3)-dependent cell lines, the myelomonocytic cell line WEHI-3, and a large proportion of primary myeloid and erythroid colony-forming cells. Spleen- and bone marrow-derived 114/A10-positive cells were shown to selectively proliferate in vitro in response to pokeweed mitogen-stimulated spleen cell-conditioned medium or recombinant IL-3. Western blot analysis indicated that the antigen recognized by mAb 114/A10 has a mean relative molecular mass of approximately 150,000, although it is extremely heterogeneous in nature, and differs greatly in size range among different cell lines.