A novel immunocytochemistry technique to measure hsp70 induction in L929 cells exposed to cadmium chloride.

The heat shock response has been suggested as a potential biomarker in toxicology. A vast amount of stimuli have been shown to induce heat shock proteins and new techniques to measure the response are constantly being assessed. In this study we use a novel immunocytochemistry technique to measure heat shock protein 70 (hsp70) induction in L929 cells exposed to cadmium chloride. Hsp70 induction was quantifiably measured using a soluble coloured substrate and qualitatively measured using a coloured substrate that precipitated at the location of hsp70. Using the insoluble coloured substrate hsp70 was identified predominantly within the cytoplasm of control cells. At intermediate cadmium concentrations hsp70 was observed to translocate to the nucleus. At these intermediate concentrations a heterogeneous heat shock response was observed. At lethal concentrations a strong heat shock response was observed with a widespread cellular response. This study demonstrates the potential of this immunocytochemistry technique to measure toxicological effects in cells by identifying the response quantitatively and qualitatively.     

Analysis of stress-induced gene expression in fish cell lines exposed to heavy meals and heat shock

We have examined the effect of heavy metals on the expression of two major groups of stress-induced proteins in fish cell lines: the 70 kDa heat-shock proteins (hsp70) and metallothioneins (MTs). The rainbow trout hepatoma (RTH) cell line synthesized the hsp70 protein in response to zinc and heat shock, while chinook salmon embryonic (CHSE) cells synthesized this protein in response to these inducers, as well as cadmium. The synthesis of this 70 kDa protein was correlated with the accumulation of hsp70 mRNA as measured by hybridization to a trout hsp70 gene probe. Heavy metals also induced the synthesis of MT in RTH cells. However, heat shock did not result in induction of MT and its mRNA. Unlike RTH cells, CHSE cells did not synthesize MT following exposure to cadmium or zinc. When these cells were treated with 5-azacytidine prior to heavy metal treatment, accumulation of MT mRNA was observed. Northern blot analysis of total RNA from 5-azacytidine treated CHSE cells, using a trout MT (tMT-B) cDNA probe, indicated that the time-course of induction and the maximal level of MT mRNA accumulation in response to cadmium and zinc paralleled that observed in RTH cells. Copper and dexamethasone were ineffective in inducing MT mRNA in 5-azacytidine-treated CHSE cells. These results indicate that MT is specifically induced in response to heavy metal treatment, whereas the synthesis of hsp70 appears to be a general stress response. Furthermore, MT is differentially regulated by heavy metals and dexamethasone in these cell lines and the expression of MT is cell-type-specific.     

Analysis of Metal-Regulated Metallothionein and Heat Shock Gene Expression in HeLa-Derived Cadmium-Resistant Cells

The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd²⁺and Zn²⁺exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd²⁺-exposed H454 cells at levels comparable to the ones present in Cd²⁺-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Activation of heme oxygenase and heat shock protein 70 genes by stress in human hepatoma cells

Effects of various stresses were examined on the accumulation of mRNA for microsomal heme oxygenase and a heat shock protein, hsp70, in three human hepatoma cell lines. By heat shock, hsp70 mRNA was induced in all three hepatoma lines, Hep G2, Hep 3B and Hep G2f, while heme oxygenase mRNA was increased only in Hep 3B. Time-courses of the heat shock induction of both mRNAs in Hep 3B were similar. Arsenite caused induction of both mRNAs in all three cell lines, while cadmium increased them in Hep G2 and Hep 3B, but not in Hep G2f cells. These findings suggest that, although both hsp70 and heme oxygenase are heat shock proteins, the mode of induction of mRNAs for these proteins is different.     

A comparison of heat shock protein genes from cultured cells of the cabbage armyworm, Mamestra brassicae, in response to heavy metals

Heat shock protein (HSP) genes, hsp90, hsp70, hsc70, hsp20.7, and hsp19.7, were cloned and sequenced from cultured cells of the cabbage armyworm, Mamestra brassicae. Analyses of the cDNA sequences revealed open reading frames of 2,151, 1,914, 1,962, 540, and 465 bp in lengths, which encode proteins with calculated molecular weights of 82.5, 69.9, 71.6, 20.7, and 19.7 kDa, respectively. An increased expression was observed in all five genes after exposure to a high temperature. The induction of gene expression was not observed during a low temperature exposure, but was observed when the cells were recovered at ambient temperature. Expression of hsp90, hsp70, and hsp20.7 was induced after exposure to 2 microM of cadmium, while the minimum cadmium concentration for induction of hsp19.7 was 5 microM. The induction of hsp90 expression was somewhat masked by basal levels of expression. Only hsp20.7 expression was induced by exposure to copper. Lead did not induce expression of any of the HSP genes tested. Cadmium-induced up-regulation of hsp70 expression was lasted longer than heat-induced one. These results suggest that hsp70 could be useful to assess the cellular distress or injury induced by cadmium.     

Differential heat shock gene hsp70-1 response to toxicants revealed by in vivo study of lungs in transgenic mice

Members of heat shock proteins (Hsp70) family have been considered to respond to a large variety of stressful conditions. But it was suggested that, in pulmonary cells, Hsp response depends more closely on the type of stimulus. The lungs are critical organs potentially subjected to air pollution affecting respiratory function and, therefore, these organs are of particular interest with regard to the stress response. To investigate the stress dependence of Hsp70 response in lungs, we created transgenic mice where the firefly luciferase reporter gene is under the control of the murine hsp70-1 promoter and exposed them to different sublethal toxic conditions. For each condition, the level of transgene induction and pulmonary toxicity were assessed. We found that hsp70-1 promoter was stimulated by heat shock and cadmium but not by ozone, paraquat, and parathion, even if these chemicals induced respiratory distress and lung inflammation. Similar observations were made when expression of the endogenous hsp70-1 gene was analyzed, indicating that our transgenic model was accurately detecting hsp70-1 induction. Thereby, it appeared that hsp70-1 response is selective and depends on signaling pathways triggered by the toxicants rather than by their pathologic toxicity per se. Furthermore, because all the chemicals used in our study have been previously described to increase the level of oxidative stress, it indicates that there is no direct and simple correlation between hsp70-1 response and the level of oxidative stress, but more specific oxidative patterns should be involved in Hsp regulation.     

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.     

Examination of cadmium-induced expression of the small heat shock protein gene, hsp30, in Xenopus laevis A6 kidney epithelial cells

Cadmium is a highly toxic environmental pollutant that has been classified as a human carcinogen. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. In the present study, we have characterized the effect of cadmium on the accumulation of the small heat shock protein (HSP), HSP30, in Xenopus laevis A6 kidney epithelial cells. Incubation of A6 cells with cadmium chloride induced the accumulation of HSP30 protein and hsp30 mRNA. While HSP70 protein and hsp70 mRNA accumulation were also induced, the relative levels of actin remained relatively unaffected. Elevated levels of HSP30 were detected in cells undergoing prolonged exposure of cells to cadmium chloride or in cells recovering from cadmium chloride treatment. Immunocytochemical analysis of cadmium chloride-treated A6 cells revealed HSP30 accumulation primarily in the cytoplasm in a punctate pattern supplemented with larger HSP30 staining structures. Also, HSP30 co-localized with the F-actin cytoskeleton at higher cadmium chloride concentrations. The combination of mild heat shock temperatures plus cadmium chloride concentrations employed in this study resulted in a synergistic accumulation of HSP30 protein and hsp30 mRNA. Finally, in contrast to heat shock, prior exposure of Xenopus A6 cells to cadmium chloride treatment, sufficient to induce the accumulation of HSPs, did not protect the cells against a subsequent thermal challenge.     

70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Comparison of the effect of heat shock factor inhibitor, KNK437, on heat shock- and chemical stress-induced hsp30 gene expression in Xenopus laevis A6 cells

In this study, we compared the effect of KNK437 (N-formyl-3, 4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat shock and chemical stressor-induced hsp30 gene expression in Xenopus laevis A6 kidney epithelial cells. Previously, KNK437 was shown to inhibit HSE-HSF1 binding activity and heat-induced hsp gene expression. In the present study, Northern and Western blot analysis revealed that pretreatment of A6 cells with KNK437 inhibited hsp30 mRNA and HSP30 and HSP70 protein accumulation induced by chemical stressors including sodium arsenite, cadmium chloride and herbimycin A. In A6 cells subjected to sodium arsenite, cadmium chloride, herbimycin A or a 33 degrees C heat shock treatment, immunocytochemistry and confocal microscopy revealed that HSP30 accumulated primarily in the cytoplasm. However, incubation of A6 cells at 35 degrees C resulted in enhanced HSP30 accumulation in the nucleus. Pre-treatment with 100 microM KNK437 completely inhibited HSP30 accumulation in A6 cells heat shocked at 33 or 35 degrees C as well as cells treated with 10 microM sodium arsenite, 100 microM cadmium chloride or 1 microg/mL herbimycin A. These results show that KNK437 is effective at inhibiting both heat shock- and chemical stress-induced hsp gene expression in amphibian cells.