Astatine (211At) as a therapeutic radionuclide. The plasma: Blood cell distribution in vitro

Therapy of carcinoma of the thyroid may include the use of the radionuclide ¹³¹I, which localizes to thyroid tissue. In considering the use of another halogen, the α particle emitting radionuclide astatine, ²¹¹At, there is also the requirement that it too can be taken up by the thyroid. However, in view of its short half-life (7.2 h) it is important that its transport in the blood is not a factor likely to render it less available. For example, retention of ²¹¹At by red cells may retard its uptake by the thyroid. This in vitro in-vestigation of the partitioning of the ²¹¹At between erythrocytes and plasma indicates that it is not strongly bound by the red cells in blood.     

Improved quantification of cell survival on stromal monolayers by flow cytometric analyses

BACKGROUND: The ex vivo survival of leukemic cells maintained on bone marrow stroma is an important tool for the investigation of cell survival and leukemogenesis. Currently, ex vivo survival of leukemic cell survival is measured by coculture on stromal cell monolayers. In these assays, we postulated that two important sources of error might be introduced through either variations in flow volume or in donor stromal cells. METHODS: A previously reported coculture assay that maintains leukemic cells on bone marrow stromal cells was employed. RESULTS: We identified two means of optimizing the coculture assay. First, biologically inert beads having well-characterized fluorescent properties were added to each sample to mathematically adjust for flow-based variations in volume acquisition. The inclusion of fluorescent beads to the basic stromal cell assay showed a significantly lower coefficient of variation as compared to samples analyzed without beads or manually counted using a hemacytometer. Second, in order to minimize variability in bone marrow hematopoietic function between donors, an adherent stromal cell line known to support hematopoiesis (HS-5) was used. When normal human donor stromal cells were used, variability in the survival of leukemic cells was observed on stromal cells derived from different donors. In contrast, statistically significant variability in survival of leukemic cells was not seen on HS-5 monolayers. Finally, we demonstrate that patient-derived leukemic samples may be examined for cell survival using these modifications. CONCLUSIONS: The novel use of fluorescent beads and a hematopoietic-supportive stromal cell line together makes the quantification of stroma-supported cell survival more reproducible, accurate, and amenable to patient-derived samples. These improvements in flow cytometry-based cell quantification are an important step in establishing a role for stromal cell assays in the study of leukemia biology and therapy.     

The effects of benzamide ADP-ribosyl transferase inhibitors on cell survival and DNA strand-break repair in irradiated mammalian cells

We have recently shown that 3-acetamidobenzamide (3-AAB), a highly effective inhibitor of ADP-ribosyl transferase (ADPRT), can act as a post-irradiation (electrons) sensitizer on the mouse lymphoma cell lines L5178Y R and S. We have now shown that this compound sensitizes human derived skin fibroblasts but to a lesser extent. Fibroblasts derived from normal, Friedreich's ataxia, and ataxia-telangiectasia individuals were equally sensitized by 3-AAB to electron radiation. 3-AAB was also effective in sensitizing the mouse lymphoma lines to fast neutron irradiation. In addition DNA strand break repair was retarded as had been found after electron irradiation. 3-Nitrobenzamide is structurally a potentially dual action radiation sensitizer with electron affinic and ADPRT inhibitory properties. It is a weaker inhibitor of ADPRT compared to 3-AAB, and results in a smaller sensitization of mouse lymphoma cells in air. However, a much greater sensitization is achieved in anoxia. This greater sensitization appears to be a synergistic rather than an additive combination of its electron affinic and ADPRT inhibitory properties.     

The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells.

Survival and induction of DNA double-strand breaks were studied in cells of Saccharomyces cerevisiae irradiated under oxic or anoxic conditions with 30 MeV electrons. A linear relationship between DNA double-strand breakage and dose was found in both cases. The o.e.r.-value for colony forming ability was found to be 1.9 +/- 0.2, whereas the o.e.r.-value for DNA double-strand breakage was 3.0 +/- 0.1. These results are not inconsistent with the idea that DNA double-strand breaks are involved in killing of yeast cells. The frequency of induction of DNA double-strand breaks was found to be 0.74 x 10(-11) double-strand breaks per g/mol per Gy when cells were irradiated under oxygen and 0.24 x 10(-11) double-strand breaks per g/mol per Gy under nitrogen.     

Enhanced heterodimerization of Bax by Bcl-2 mutants improves irradiated cell survival

B Cell Lymphoma-2 (Bcl-2) protein suppresses ionizing radiation-induced apoptosis in hemato-lymphoid system. To enhance the survival of irradiated cells, we have compared the effects and mechanism of Bcl-2 and its functional variants, D34A (caspase-3 resistant) and S70E (mimics phosphorylation on S70). Bcl-2 and its mutants were transfected into hematopoietic cell line and assessed for cell survival, clonogenicity and cell cycle perturbations upon exposure to ionizing radiation. The electrostatic potential of BH3 cleft of Bcl-2/mutants and their heterodimerization with Bcl-2 associated X protein (Bax) were computationally evaluated. Correspondingly, these results were verified by co-immunoprecipitation and western blotting. The mutants afford higher radioprotective effect than Bcl-2 in apoptotic and clonogenic assays at D₀ (radiation dose at which 37 % cell survival was observed). The computational and functional analysis indicates that mutants have higher propensity to neutralize Bax protein by heterodimerization and have increased caspase-9 suppression capability, which is responsible for enhanced survival. This study implies potential of Bcl-2 mutants or their chemical/peptide mimics to elicit radioprotective effect in cells exposed to radiation.     

Release of mitotic descendants by giant cells from irradiated Burkitt's lymphoma cell line.

Polyploid giant cells are produced as part of the response of p53 mutant Burkitt's lymphoma cell lines to high doses of irradiation. Polyploid giant cells arise by endo-reduplication in the first week after a single 10 Gray dose of irradiation. Within the giant cells a sub-nuclear structure is apparent and within this, sub-nuclear autonomy is evident, as displayed by independent nuclear structure and DNA replication in different parts of the nucleus. The majority of these cells soon die as apoptotic polykaryons. However, approximately 10-20% of giant cells remain viable into the second week after irradiation and begin vigorous extrusion of large degraded chromatin masses. During the second week, the giant cells begin to reconstruct their nuclei into polyploid 'bouquets', where chromosome double-loops are formed. Subsequently, the bouquets return to an interphase state and separate into several secondary nuclei. The individual sub-nuclei then resume DNA synthesis with mitotic divisions and sequester cytoplasmic territories around themselves, giving rise to the secondary cells, which continue mitotic propagation. This process of giant cell formation, reorganization and breakdown appears to provide an additional mechanism for repairing double-strand DNA breaks within tumour cells.     

At least two separate gene clusters are involved in albicidin production by Xanthomonas albilineans.

Transposon mutagenesis was used to obtain mutations affecting production of the toxin albicidin in Xanthomonas albilineans, which causes leaf scald disease of sugarcane and is also pathogenic to corn. Transposon Tn5-gusA inserted randomly into genomic DNA of X. albilineans Xa23R1 at a frequency of 10(-4) to 10(-5) per recipient after conjugal transfer from Escherichia coli. Fifty prototrophic mutants defective in albicidin production were isolated from 7,100 Tn5-gusA insertional derivatives tested for toxin production by an antibiosis bioassay. EcoRI fragments containing Tn5 flanking sequences from two mutants (AM15 and AM40) were cloned and used to probe a wild-type Xa23R1 DNA library by colony hybridization. Nine cosmids showed homology to the AM15 probe, and six showed homology to the AM40 probe. Four cosmid clones hybridized to both probes. Forty-five of the 50 defective mutants were restored to albicidin production with two overlapping cosmid clones. Restriction mapping showed that these mutants span a genomic region of about 48 kb. At least one other gene cluster is also involved in albicidin production in Xa23R1. DNA fragments from the 48-kb cluster proved to be very specific to X. albilineans. Some mutants affected in albicidin production retain their ability to colonize sugarcane cultivated in vitro.     

The mechanism of sensitivity to phleomycin in growing Escherichia coli cells.

Stationary-phase Escherichia coli B cells transferred to new growth medium are initially resistant to net DNA breakage by low concentrations of phleomycin, and become sensitive as DNA replication commences. From studies with inhibitors of various stages of the DNA replication cycle it is evident that it is not DNA synthesis itself that is required for induction of DNA breakage by phleomycin, but events associated with the initiation of DNA replication. Termination of replication in the absence of further initiaiton results in resistance to phleomycin. The cellular change responsible for changes in sensitivity to phleomycin could be the attachment of the bacterial chromosome to the cell membrane at initiation and detachment on termination of replication, suggesting an alteration in the balance between cellular DNA breakage and repair processes for membrane-associated compared with non-membrane-associated DNA.     

Failure of 5-thio-D-glucose to alter cell survival in irradiated or unirradiated EMT6 tumors

The antineoplastic effects of the glucose analog 5-thio-D-glucose (5TG) were tested using EMT6 mouse mammary tumors in vivo and EMT6 cells in cell culture. In vitro, 5TG selectively killed hypoxic EMT6 cells. However, administration of 5TG to mice bearing EMT6 tumors produced no significant toxicity to the cells of unirradiated tumors and did not alter the survival of cells in irradiated tumors. Fasting the mice to lower blood glucose concentrations before administration of 5TG increased the toxicity of the drug to the mice, but did not allow more efficacious treatment of the tumors. The data provided no evidence that 5-thio-D-glucose can be used effectively for the treatment of solid tumors, either as a cytotoxic agent or as a radiosensitizer.     

Growth of mouse fibroblasts in the presence of irradiated and lyophilized monolayers of the same type of cells

Certain cells, such as 3T3 mouse embryo fibroblasts, are inhibited from dividing when they grow to a characteristic cell density on a surface in tissue culture. We asked whether the inhibition of cell division could be attributed to the inert chemical composition of neighboring cells, that is, whether the residues of lyophilized cells retained the ability to inhibit the division of normal cells. In addition, we wanted to know whether cells in which DNA synthesis was imparied by irradiation would retain the capacity to effectively inhibit normal cells. To answer these questions, confluent and non-confluent layers of 3T3 cells were prepared in tissue culture dishes and the cells were either lyophilized or irrariated in situ. Fresh 3T3 cells were then added to these prepared layers and their growth was followed using radioactive label. There was no growth of added cells on the confluent monolayers of either untreated or irradiated cells. Growth was unimpeded on the monolayers of lyophilized cells. When cells were added to non-confluent cultures of either normal or irradiated cells the added cells grew until they had covered the remaining surface of the culture dish and had come into contact with the pre-existing cells. In the discussion, consideration is given to the role of available surface over which the cells can spread as well as to the possible interactions between neighboring cells.     

Stimulation of mononuclear cell binding to human endothelial cell monolayers by thrombin

The common occurrence of fibrin deposits in chronic inflammatory lesions suggests a possible role for thrombin in the mobilization of mononuclear cell infiltrates. For this reason, the effect of thrombin on the binding of mononuclear cells to endothelial cells (EC) was investigated. Incubation of confluent monolayers of human umbilical vein endothelial cells with thrombin markedly enhanced EC adhesiveness for both T lymphocytes and U937 cells (a monocyte-like cell line) in a time- and dose-dependent fashion. This effect was EC specific: 1) treatment of the T cells or the U937 cells with thrombin did not stimulate their adherence to EC, and 2) treatment of human foreskin fibroblasts with thrombin did not stimulate their inherently low adhesiveness for T cells. Fixation of EC monolayers with paraformaldehyde after pre-incubation with thrombin did not affect the increased adhesiveness for T cells. mAb against the LFA-1 antigen (mAb 60.3 (anti-CD18) or mAb TS1/22 (anti-CD11a), which inhibit the binding of T cells to unstimulated EC, failed to block the increased adhesion induced by thrombin, indicating that the increased binding induced by thrombin is similar to that induced by IL-1 and TNF, which showed similar resistance. These results suggest that thrombin may have a role in the extravascular emigration of mononuclear cells from post-capillary venules by virtue of its ability to stimulate the adhesiveness of EC for both lymphocytes and monocytes.     

The survival rate of irradiated cells as a solution of a stochastic differential equation. A general description

The survival rate function is described as a solution of some stochastic differential equation. Two main reasons for the formation of dispersion of irradiated cell survival were analysed: accidental changes in reactivity of individual cells during irradiation and differences in reactivity of individual cells within the exposed ensemble. It was shown that in both cases dose response of the survival dispersion was different.     

Molecular counting of low-density lipoprotein particles as individuals and small clusters on cell surfaces.

We employ the intensely fluorescent analogue diI-LDL (Barak, L. S., and W. W. Webb, 1981, J. Cell Biol. 90:595-604) as a counting marker to determine the numbers of LDL-receptor complexes that are contained in clusters on the surfaces of human fibroblasts and human epidermoid carcinoma cells. The application of quantitative digital intensified video optical microscopy allows the measurement of the fluorescence power collected from individual fluorescent spots on a cell with sufficient accuracy that the number of optically unresolved particles producing the fluorescence in the spot can be estimated. We demonstrate that isolated individual diI-LDL particles are detected on the surface of all cells investigated. Analysis of the LDL cluster size distributions on the various cell lines shows clear differences that correlate with efficiency of LDL metabolism. We find that normal fibroblasts (GM3348) have LDL-receptor complex populations dominated by large cluster sizes (greater than 4 LDL), while internalization-deficient J.D. mutant fibroblasts (GM2408A) and epidermoid carcinoma cells (A-431) show predominantly small clusters (1-3 LDL). No evidence for large-scale ordering or "superclustering" of clusters is found.     

Binding of progesterone to cell surfaces of human granulosa-lutein cells

Progesterone is produced by granulosa cells under the influence of luteinizing hormone. Nuclear progesterone receptors have been found in rat granulosa cells. Human granulosa-lutein cells rapidly respond to progesterone with an increase in intracellular calcium suggesting the existence of a nongenomic mechanism. This study was conducted to determine whether binding of progesterone to granulosa cells could occur at the membrane. Granulosa cells were obtained from an in vitro fertilization program and examined immunohistochemically with an antiserum to membrane progesterone receptors. Approximately 14-70% of freshly harvested or cultured granulosa cells of six patients showed a positive reaction to the antiserum, limited to the cell membrane. Western blot analysis of homogenates of granulosa cells and a granulosa cell tumour confirmed the presence of progesterone receptors A, B and C and low amounts of a putative membrane receptor. These results demonstrate that the plasma membranes of human granulosa cells possess binding components for progesterone which may be involved in its nongenomic mechanism of action.