Effect of protective agents on amount and repair of sublethal freeze--thaw damage in mammalian cells.

Glycerol, DMSO, and HES are able to reduce by a factor of 2 the sublethal damage produced in mammalian cells after one freeze-thaw cycle. When sublethal freeze-thaw damage is already present, DMSO and HES are able to prevent about half of this damage from becoming lethal when a second freeze-thaw cycle is applied. Glycerol is only able to do this if dilution shock is avoided by thawing the cells into medium containing glycerol. The cells can repair 100% of this sublethal damage and do so in 2–3 hr at 37 °C in suspension. The data imply that the sites protected by DMSO, HES, and glycerol are the same as the sites repaired by the cells. The results also suggest that cells stop progressing in the cell cycle while repairing sublethal freeze-thaw damage.     

Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells.

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.     

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.     

Factors affecting the repair of sublethal freeze-thaw damage in mammalian cells: II. The effect of ouabain

Mammalian cells were able to repair sublethal damage sustained during exposure to freeze-thaw conditions if they were incubated at 37 °C during the repair period. Repair was also observed when the cells were incubated at 37 °C in medium containing 10^?4m ouabain but this was not the case with 10^?3m ouabain. Cells exposed to either 10^?3 or 10^?4m ouabain before freezing and thawing showed reduced survival indicating the requirement for the prior operation of the (Na⁺ ? K⁺) — ATPase system to avoid additional lethal damage.     

Radiobiology of stem cells in intestinal epithelium. Effect of single and multiple preliminary sublethal irradiation of mice on the dose dependence of the survival of stem cells in small intestine epithelium

After a single or three-fold whole body irradiation of mice with a dose of 4 Gy and the time interval for the proliferation to be restored (5 days or 3 weeks) the survival curve for stem cells of small intestine epithelium with regard to radiation dose was the same as that for non-preirradiated mice. This indicated that the proliferative potential of stem cells in these experimental conditions was not reduced.     

Effect of membrane fatty acid substitution and temperature on repair of sublethal damage in mammalian cells

Repair of sublethal radiation damage (SLD) has been investigated as a function of temperature in mouse fibroblast LM cells with different membrane lipid composition. Rigidification or fluidization of the cellular membranes was accomplished by incorporation of myristic acid and arachidonic acid, respectively, in the phospholipids of the membranes. The SLD repair after radiation was essentially the same for the cells with the more rigid (saturated fatty acid) membranes and the cells with the more fluid (polyunsaturated fatty acid) membranes. This observation was made for repair at 37 degrees C as well as for repair at hypothermic temperatures. Incorporation of polyunsaturated fatty acid protected the cells against hypothermic death. These experiments demonstrate that although membranes are likely targets for cell killing by low temperature treatments, membrane lipids are probably not involved in the repair of sublethal radiation damage. It must be concluded that neither the degree of polyunsaturation of the lipids nor the degree of fluidity of the membrane is important for radiation-induced killing of mammalian cells.     

Factors affecting the repair of sublethal freeze-thaw damage in mammalian cells : I. Suboptimal temperature and hypoxia

The repair of sublethal freeze-thaw damage has been demonstrated by exposing cells to two freeze-thaw cycles separated by various times at 37 °C. The influence of suboptimal temperatures and hypoxia on this repair process was investigated. Repair was found to be temperature sensitive, decreasing as the temperature was lowered from 37 °C, and was virtually nonexistent at 5 °C. Lowering the oxygen concentration in the medium to 6.6 μM or to 0.66 μM had no significant effect on repair. The repair system then appears to involve an enzymatic process which is not greatly dependent on the oxygen concentration in the range of 277 (aerobic) to 0.66 μM O₂ in the medium.     

Lethal and sublethal effects of marine sediment extracts on fish cells and chromosomes

The cost of conducting conventional chronic bioassays with every potentially toxic compound found in marine ecosystems is prohibitive; therefore short-term toxicity tests which can be used for rapid screening were developed. The tests employ cultured fish cells to measure lethal, sublethal or genotoxic effects of pure compounds and complex mixtures. The sensitivity of these tests has been proven under laboratory conditions; the following study used two of these tests, the anaphase aberration test and a cytotoxicity assay, under field conditions. Sediment was collected from 97 stations within Puget Sound, Washington. Serial washings of the sediment in methanol and dichloromethane yielded an organic extract which was dried, dissolved in DMSO and incubated as a series of dilutions with rainbow trout gonad (RTG-2) cells. The toxic effects of the extract were measured by examining the rate of cell proliferation and the percentage of damaged anaphase figures. Anaphase figures were considered to be abnormal if they exhibited non-disjunctions, chromosome fragments, or chromosome bridges. A second cell line (bluegill fry, BF-2) was also tested for cell proliferation and was included because, unlike the RTG-2 cell line, it contains little or no mixed function oxygenase activity. Of 97 stations tested, 35 showed no genotoxic activity, 42 showed high genotoxic activity (P.01) and the remainder were intermediate. Among the toxic sites were several deep water stations adjacent to municipal sewage outfalls and four urban waterways contaminated by industrial and municipal effluents. Extracts from areas that showed genotoxic effects also inhibited cell proliferation and were cytotoxic to RTG-2 cells. Few effects were noted in the MFO deficient BF-2 cells. Short term in vitro tests provide aquatic toxicologists with a versatile and cost effective tool for screening complex environments. Through these tests one can identify compounds or geographic regions that exhibit high cytotoxic or genotoxic potential.     

The rate of deoxyribonucleic acid synthesis by cultured Chinese-hamster ovary cells. An application of isotope-dilution analysis.

The rate of DNA synthesis is exponentially growing cells was determined by isotopedilution analysis of the incorporation of [me-3H]thymidine. Thymidine concentrations greater than 7 micrometer were used so that the rate-limiting step governing incorporation would be at the level of DNA polymerase rather than at the level of thymidine kinase [Sjostrom & Forsdyke (1974) Biochem. J. 138, 253-262]. In early exponential phase the rate determined by isotope-dilution analysis closely correlated with the rates calculated either from growth curves or from known cell-cycle parameters. However, in late-exponential phase the rate calculated from the growth curve was less than that determined by isotope-dilution analysis. We conclude that, under certain conditions, the pool-corrected rate of incorporation of [me-3H]thymidine, as determined by isotope-dilution analysis, can accurately reflect the rate of DNA synthesis. Discrepancies between the observed rate of DNA synthesis and increase in cell number could reflect an exponential degeneration of post-S-phase cells.     

Synthesis and secretion of active lipoprotein lipase in Chinese-hamster ovary (CHO) cells.

Cultured Chinese-hamster ovary cells (CHO cells) were found to produce and secrete a lipase, which was identified as a lipoprotein lipase by the following criteria. Its activity was stimulated by serum and apolipoprotein CII, and was inhibited by high salt concentration. The lipase bound to heparin-agarose and co-eluted with 125I-labelled bovine lipoprotein lipase in a salt gradient. A chicken antiserum to bovine lipoprotein lipase inhibited the activity and precipitated a labelled protein of the same apparent size as bovine lipoprotein lipase from media of CHO cells labelled with [35S]methionine. The lipase activity and secretion were similar in growing cells and in cells that had reached confluency. Hence, lipoprotein lipase appears to be expressed constitutively in CHO cells and is not linked to certain growth conditions, as in pre-adipocyte and macrophage cell lines. At 37 degrees C, but not at 4 degrees C, heparin increased the release of lipase to the medium 2-4-fold. This increased release occurred without depletion of cell-associated lipase activity, suggesting that heparin enhanced release of newly synthesized lipase.     

Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.