diamide and sodium-arsenite

Thermotolerance (TT) induced by sodium arsenite (A-TT: 100 microM, 1 h, 37 degrees C) was compared to heat-induced thermotolerance (H-TT: 15 min, 44 degrees C) using HeLa S3 cells. All four pretreatments led to comparable levels of thermotolerance and also induced resistance to arsenite-, ethanol-, and diamide-induced toxicity (clonogenic ability). Stress-induced expression of the major heat shock proteins (hsp27, hsc70(p73), hsp70(p72), and hsp90) was generally highest in H-TT cells and lowest in A-TT cells. Interestingly, the four types of TT cells showed distinct differences in certain aspects of resistance against thermal protein damage. Thermal protein denaturation and aggregation determined in isolated cellular membrane fractions was found to be attenuated when they were isolated from H-TT and A-TT cells but not when isolated from E-TT and D-TT cells. The heat resistance in the proteins of the membrane fraction corresponded with elevated levels of hsp70(p72) associated with the isolated membrane fractions. In the nuclear fraction, only marginal (not significant) attenuation of the formation of protein aggregates (as determined by TX-100 (in)solubility) was observed. However, the postheat recovery from heat-induced protein aggregation in the nucleus was faster in H-TT, E-TT, and D-TT cells, but not in A-TT cells. Despite the fact that elevated levels of hsp27, hsp70(p73), and hsp70(p72) were found in the TX-100 insoluble nuclear fraction derived from all TT cells, no correlation was found with the degree of resistance in terms of the accelerated recovery from nuclear protein aggregation. The only correlation between accelerated recovery from nuclear protein aggregates was that with total cellular levels of hsp27. The data indicate that heat-induced loss of clonogenic ability may be a multitarget rather than a single target event. A threshold of damage may exist in cells after exposure to heat; multiple sets of proteins in (different compartments of) the cell need to be damaged before this threshold is exceeded and the cell dies. As a consequence, stabilization of only one of these sets of proteins is already sufficient to render cells thermotolerant at the clonogenic level.

Topics: Arsenites; Diamide; Ethanol; Heat-Shock Proteins; HeLa Cells; Humans; Protein Conformation; Protein Denaturation; Proteins; Sodium Compounds; Temperature

Erythrocytes treated with various chemical agents for 1 h at 37 degrees C showed resistance to a subsequent 1 h heat treatment at 53 degrees C. Maximal thermotolerance was observed 6 h after 3 mM DNP and 0.03 mM disulfiram treatment and 4 h after diamide exposure at 0.3 mM. Our results suggest that chemically induced thermotolerance to heat treatment in erythrocytes was similar to heat-induced thermotolerance.

Topics: 2,4-Dinitrophenol; Acclimatization; Animals; Arsenic; Arsenites; Diamide; Dinitrophenols; Disulfiram; Erythrocytes; Hot Temperature; In Vitro Techniques; Sodium Compounds; Sulfhydryl Reagents; Swine

The cellular heat shock response leads to the enhanced synthesis of a family of heat shock proteins and the development of thermotolerance. In CHO cells, however, heat shock also leads to enhanced synthesis of a 50 kD glycoprotein and elevated activity of N-acetylgalactosaminyltransferase (GalNAcT). In this study we showed increased GalNAcT activity during thermotolerance expression in all of five mammalian cell lines included in the study. However, there was no simple correlation between cellular heat sensitivity of unheated control cells and basal levels of GalNAcT activity, measured toward the same exogenous acceptor apomucin. Although GalNAcT was elevated in thermotolerant cells, GalNAcT activity itself did not exhibit thermotolerance in terms of reduced sensitivity to heat inactivation. The increase in GalNAcT activity after heating was similar in exponentially growing and plateau-phase cultures and was inhibited neither by cycloheximide nor actinomycin D. However, the inhibitors by themselves also increased GalNAcT activity in unheated control cells. Chemical inducers of thermotolerance (arsenite and diamide) increased GalNAcT activity, but the increase was modest when compared to that following hyperthermia. In addition to GalNAcT, two other glycosyltransferases with specificity for O-glycans, alpha 1,2-fucosyltransferase and alpha 2,6-sialyltransferase, also showed increased activity after hyperthermia and during thermotolerance development. Together with previously published data, these results support the hypothesis that heat-induced activation of O-glycan-specific glycosyltransferases plays a physiological role in the cellular heat shock response and in thermotolerance development.

Topics: Animals; Arsenic; Arsenites; beta-D-Galactoside alpha 2-6-Sialyltransferase; Breast Neoplasms; Cell Line; Cricetinae; Cycloheximide; Dactinomycin; Diamide; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Galactosyltransferases; Heat-Shock Proteins; Hot Temperature; Humans; Kinetics; N-Acetylgalactosaminyltransferases; Sialyltransferases; Sodium Compounds; Tumor Cells, Cultured

Thermotolerance and synthesis of heat shock proteins are induced in cells in response to a variety of environmental stresses. We examined the suggestion of Hightower (1980) that modifications of intracellular proteins may be the triggering event that induces heat shock protein synthesis and thermotolerance. We did so by modifying cellular proteins, using diamide, a sulfhydryl oxidizing agent, and dithio-bis (succinimidyl propionate), an agent that cross-links bifunctional amino groups. Both of these agents induced heat shock proteins and thermotolerance in CHO (HA-1) cells. Furthermore, we observed cross-resistance and self-tolerance with three seemingly unrelated stimuli (diamide, heat, and sodium arsenite). This observation suggests that the induction of protective responses to these stimuli is mediated by a common mechanism. The results support the hypothesis that production of abnormal proteins by various stresses induces the stress responses as well as tolerance.

Topics: Animals; Arsenic; Arsenites; Azo Compounds; Cell Line; Cricetinae; Diamide; Dithiothreitol; Glutathione; Heat-Shock Proteins; Hot Temperature; Proteins; Sodium Compounds; Stress, Physiological

The mechanism by which chemical inducers of the stress response inhibit protein synthesis was examined. All the chemicals tested principally inhibit the initiation phase of translation. Covalent modification of the initiation factor proteins does not constitute a common mechanism. Eukaryotic initiation factor (eIF)-2 alpha phosphorylation is moderately to strongly induced by Na arsenite and diamide, but only slightly to imperceptibly affected by iodoacetamide, azetidine carboxylic acid, and canavanine. eIF-4B dephosphorylation does not occur in any case. The only consistent change detected is the hyperphosphorylation of the 28,000 Da heat stress protein. These results indicate that these diverse chemicals, all of which enhance the transcription of the stress mRNAs, do not inhibit translation by a common, recognized mechanism; it is likely that several distinct pathways leading to inhibition exist.

Topics: Arsenic; Arsenites; Azetidinecarboxylic Acid; Azetines; Azo Compounds; Canavanine; Diamide; HeLa Cells; Humans; Iodoacetamide; Iodoacetates; Neoplasm Proteins; Peptide Chain Initiation, Translational; Protein Biosynthesis; Sodium Compounds; Stress, Physiological