DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this "contact effect" to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.     

Lysis of small cell carcinoma of the lung tumor cell lines by gamma interferon-activated allogeneic peripheral blood mononuclear cells: abrogation of killing by pretreatment of tumor cells with gamma interferon

Summary Interferon has been shown to enhance the ability of nonspecific cytotoxic mononuclear cells to lyse some, but not all, tumor cells. We have examined the effect of recombinant human gamma interferon (rIFN) on the cell-mediated cytolysis of tumor target cells derived from continuously cultured lines of small cell carcinoma of the lung (SCCL). Cells from the SCCL lines DMS 44, 53, 79, 92, and 406 were labeled with ⁵¹Cr and incubated with normal and rIFN-stimulated peripheral blood mononuclear cells for 18 h at 37 °C and tumor cell lysis estimated by measuring ⁵¹Cr release. Although cells from certain SCCL lines were good targets for cell mediated cytotoxicity, susceptibility to lysis was heterogenous among the different SCCL lines. DMS 406 and 79 were, on average, maximally lysed, while DMS 44, 53, and 92 showed less susceptibility to lysis by either control or rIFN-stimulated effector cells. In addition, although pretreatment with rIFN increased the cytolytic capacity of normal peripheral blood mononuclear cells from several different donors, preincubation of the tumor cell lines with rIFN resulted in inhibition of cytolysis mediated by both control and IFN-activated effector cells. These findings suggest that although rIFN may enhance cell-mediated lysis of SCCL tumor cells, it may also decrease susceptibility to lysis.This work was supported by Grants CA 37868, CA 31888, CA31918, CA33852, and AI 19053 awarded by the National Cancer Institute and Institute of Allergy and Infectious Diseases, DHHS. Statistical assistance was provided by Therese Stukel, biostatistician at the Norris Cotton Cancer Center, Hanover, H. H. and supported by Grant CA 23108 from the National Cancer Institute     

Interacting effects of light,temperature, and nutrients on C-14 uptake of oscillatoria rubescens de candolle

The rate of C¹⁴O₂ uptake in a bacterized isolate of Oscillatoria rubescens indicates that within the ranges studied temperature caused the greatest variation followed by light intensity and nutrient concentration. The variation within interaction effects of light, temperature, and nutrients was higher than that within any other combination of interactions. High temperatures (25°C) shifted the light optimum of O. rubescens growing in low to moderate nutrient levels from 1950 lux to 800 lux.     

I. Catecholamines in developing rat lung

The development of the catecholaminergic system of lung was investigated in Sprague-Dawley rats during the perinatal period and in young adults. A radio-enzymatic procedure allowed to measure as little as 1 pg of noradrenaline or dopamine in each sample. The dopamine levels were low and independent of age. The noradrenaline levels increased significantly with the age until the 20th post-partum day. No significant differences were found between noradrenaline levels for males vs females at any age. Small differences between males and females for dopamine levels existed only near birth.     

Factors influencing DNA migration from individual cells subjected to gel electrophoresis.

Alkaline and neutral gel electrophoresis of individual mammalian cells allows detection of DNA single- and double-strand breaks, respectively. For both the alkaline and the neutral assays, lysis conditions influence how much DNA migrates, and factors in addition to DNA size play a role in migration. In particular, the tight packing of DNA in individual nuclei appears to reduce the ability to detect double-strand breaks in all of the genome. Tangling of DNA molecules is probably also responsible for the presence of "wings" associated with each nucleus after application of pulsed-field gel electrophoresis; these wings were aligned in the directions of the pulsed field, not along the resultant vector of the fields as was expected. The choice of fluorescent staining methods (propidium iodide, Hoechst 33342, or antibodies against bromodeoxyuridine) did not influence sensitivity for detecting DNA damage.     

Two distinct TL-like molecular subsets defined by monoclonal antibodies on the surface of human thymocytes with different expression on leukemia lines

Monoclonal antibodies reacting with TL-like class I antigens expressed on the surface of human thymocytes and some T leukemia lines were found to define three independent epitopic clusters, two of which could be shown to reside on serologically distinct molecular subsets by a solid-phase radioimmunoassay as well as by sequential immunoprecipitation. Both molecular subsets consist of a 49-K heavy chain associated with a beta-2 microglobulin light chain. Thymocytes expressed similar amounts of the two molecular subsets, while on T leukemia lines the amount of these two molecular subsets varied from line to line.     

Characterization of a carbocyanine derivative as a fluorescent penetration probe

The fluorescent carbocyanine dye 3,3-diheptyloxycarbocyanine [DiOC7(3)], originally described as a membrane potential probe, penetrates poorly into multicell spheroids. Since the dye is retained in the cells following spheroid disaggregation, cells can be selected from different depths within the spheroid using fluorescence-activated cell sorting. Characterization of the binding kinetics, stability, and toxicity of this probe were undertaken, and intercompared with Hoechst 33342. The optimum drug dose for achieving good separation of internal and external cells of spheroids is about tenfold lower than for Hoechst 33342, and like Hoechst, DiOC7(3) is toxic at concentrations at least tenfold higher than those required to produce a good gradient for cell separation. When cells are removed from the stain, cellular fluorescence decreases to half the initial intensity within 2 hours; however, unlike Hoechst, the carbocyanine dye does not transfer between cells.