Hyperthermia induces gene expression of heat shock protein 70 and phosphorylation of mitogen activated protein kinases in the rat cerebellum.

In-vivo heat-shock induced heat shock factor (HSF) DNA-binding activity and accumulation of heat shock protein (hsp)70 mRNA in newborn and adult rat cerebellum was studied. We identified a high basal level of c-Jun N-terminal kinase (JNK) and p38 MAP kinase phosphorylation in the cerebellum, independently of age. Hyperthermia increased JNK1, decreased JNK2 but did not modify JNK3 phosphorylation in the newborn cerebellum, whereas decreased the phosphorylation of both JNK1 and JNK3 in adult rats. During recovery from hyperthermia, JNK2 phosphorylation returned to control level in the newborn, JNK1 appeared hyperphosphorylated only in the newborn, and JNK3 in all animals. JNK2 never appeared phosphorylated in the adult cerebellum. Hyperthermia increased p38 MAP kinase phosphorylation in the cerebellum, with different trends in newborn and adult rats during recovery. Heat shock increased extracellular signal-regulated kinase phosphorylation concomitant to tyrosine kinase receptor activation (epidermal growth factor-receptor in the newborn and insulin-like growth factor-receptor in the adult cerebellum). The behavior of stress kinases may underlie a different age-related vulnerability to heat stress of the cerebellum.

Down-modulation of c-myc expression by phorbol ester protects CEM T leukaemia cells from starvation-induced apoptosis: role of ornithine decarboxylase and polyamines.

Myc is a transcriptional activator whose deregulated expression not only promotes proliferation but also induces or sensitizes cells to apoptosis. Here we demonstrate that c-myc plays a role in triggering apoptosis in CEM T leukaemia cells exposed to progressive medium exhaustion. Indeed starved cells undergo apoptosis in the presence of constitutively elevated c-myc expression and the phorbol ester, phorbol 12-miristate 13-acetate (PMA), which rescues cells from apoptosis, induces complete c-myc down-regulation. We also investigate the hypothesis that ornithine decarboxylase (ODC), a transcriptional target of c-myc, is a down-stream mediator of c-myc driven apoptosis. We demonstrate that PMA induces in starved cells an earlier and larger decrease in ODC expression (mRNA and activity) and intracellular polyamine content, compared to untreated starved cells. Moreover we show that alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzymatic activity, effectively reduces, while exogenous added polyamines enhance apoptosis in starved cells. All these data indicate that ODC and polyamines may act as facilitating factors in triggering apoptosis induced by growth/survival factors withdrawal.

Cellular signalling after in vivo heat shock in the liver.

In an experimental model of in vivo hyperthermia, we investigated the involvement of a number of signalling events in rat liver. We report that in vivo heat shock causes a powerful activation of c-Jun N-terminal kinase and p38 kinase but does not trigger poly(ADP-ribose) polymerase cleavage, a signature event of apoptosis. Among the upstream regulators of the kinases, we show that stress-activated protein kinase/extracellular signal-regulated kinase/nitrogen-activated protein kinase kinase 4 SEK1/MKK4 is not involved whereas MKK3 and/or MKK6 are activated. PAK activity displays a transient rise, whereas GCK does not change. PI3-kinase activity increases in anti-phosphotyrosine immunoprecipitates, suggesting a tyrosine kinase-dependent induction mechanism, and the co-immunoprecipitation of PI3-kinase with p60 Src kinase supports the involvement of this latter. GSK3, which may act downstream to PI3-kinase through AKT, undergoes hyperphosphorylation, thus playing a possible role in the protection from apoptosis and in the modulation of heat-shock transcription factor activity.

Cell-specific expression of heat shock transcription factors 1 and 2 in unstressed rat spinal cord.

We investigated the intracellular distribution of heat shock factors 1 and 2 (HSF1, HSF2) in rat spinal cord by immunoblotting and immunohistochemistry using selective policlonal antibodies. Our results showed that both HSF1 and HSF2 were expressed in spinal cord cells (both neurons and glia) but at different intensity and cell localization. HSF1 was unusually distributed in the perinuclear compartment of selected neurons of the gray matter while astrocytes, oligodendrocytes and ependymal cells were predominantly stained in the nucleus. HSF2 was expressed at lower levels than HSF1 and was scattered in both nucleus and cytoplasm of the motoneurons of the ventral horns while glial cells again showed a nuclear positivity. This study suggested that the different ability of neurons vs. glial cells to react against adverse conditions might well be correlated with the different constitutive localization of HSFs.

In vivo modulation of 73 kDa heat shock cognate and 78 kDa glucose-regulating protein gene expression in rat liver and brain by ethanol.

BACKGROUND: In cultured cells of various origin, ethanol induces the synthesis of 70 kDa family heat shock proteins (hsp70 family), which play a role in the protection of protein traffic and secretion, as well as in cytoskeleton organization. To assess whether ethanol also can induce such genes in vivo, we studied the behavior of hsp70, hsc73, and grp78 messenger ribonucleic acids (mRNAs) and related proteins in the liver and brain of rats acutely treated with ethanol. METHODS: Overnight fasted Sprague-Dawley rats (220-250 g) were acutely treated with a low (2 g/kg body weight) or a high (5 g/kg body weight) dose of ethanol as a 30% solution in saline or an equal volume of saline (controls) by gastric intubation. Animals were killed at various times after treatments (3-72 hr). Messenger RNA levels for different members of hsp70 family (hsp70; 73 kDa heat shock cognate, or hsc73; and 78 kDa glucose-regulating protein, or grp78) were determined by Northern blot analysis and hybridization with specific complementary deoxyribonucleic acid (cDNA) probes. The amounts of related proteins were assayed by Western blot analysis with specific antibodies. Autoradiograms and fluorograms were subjected to densitometric scanning. RESULTS: Ethanol (2 g/kg) caused a slight increase in hsc73 and grp78 mRNA levels only in the liver, without enhancing the amount of proteins. Ethanol (5 g/kg) increased the level of hsc73 and grp78 mRNAs and related proteins in the liver. In the brain, the amount of hsc73 mRNA was enhanced, but this did not change hsc73 protein. In addition, we observed an increase in cerebral grp78 transcript and related protein. Hsp70 gene was not induced in the examined tissues by either dose of ethanol. CONCLUSIONS: Hepatic and cerebral hsc73 and grp78 genes are responsive to ethanol in vivo, and their activation may signal the cell's effort to counteract the harmful action of ethanol.

Gene expression in liver after toxic injury: analysis of heat shock response and oxidative stress-inducible genes.

In the liver, CCl4 induces cell necrosis followed by regeneration. Cell injury is caused by free radical damage and may be due, at least in part, to oxidative stress and the subsequent formation of reactive oxygen intermediates (ROIs). In a rat model of acute CCl4-induced hepatic injury, we examined the expression of genes involved in cellular response to different kinds of stress, including oxidative stress (hsp 70 family, heme oxygenase), in free radical detoxification (Mn superoxide dismutase and Cu/ Zn superoxide dismutase), in iron homeostasis (H and L ferritin subunits) and in the cell cycle (c-fos, c-jun, histone H3). As an experimental approach, we first analysed the pattern of protein synthesised by liver slices in vitro. Then we studied the mechanisms regulating the expression of different genes, by analysing both mRNA steady state levels and transcription rates. Activation of the specific heat shock transcription factor (HSF) by CCl4 was also investigated. We observed that different members of the hsp70 family (hsp70, hsc73, grp78) are activated by different kinetics and are regulated mainly at the transcriptional level. Induction of the hsp70 gene occurs rapidly and transiently and is preceded by the activation of HSF DNA-binding activity. We demonstrated an increase in the steady-state levels of mRNAs for heme oxygenase, Mn and Cu/Zn superoxide dismutases and H and L ferritin subunits. However, different kinetics and regulatory mechanisms occurred with different genes. We showed that induction of c-fos and c-jun protooncogenes is the earliest event after CCl4 administration, whereas histone H3 expression peaked at 24-48 h. The results of this study are interpreted as evidence that activation of specific stress response genes is primarily related to the defence against the rapidly occurring cell damage, but may also be related to subsequent processes of tissue inflammation and cell proliferation.

Constitutive expression of heat shock proteins 70 and 90 in rat cerebellum.

Exposure to heat shock and other stressful conditions activates in cells of all organisms a specific genetic program. This enhances the synthesis of proteins with a protective role against cellular damage, called heat shock proteins (hsps). Furthermore, in the mammalian nervous system, a considerable amount of hsps is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we analysed the constitutive expression of proteins belonging to the hsp70 and hsp90 family in the rat cerebellum using immunocytochemistry with specific monoclonal antibodies. Our results showed that an intense immunostaining was evident, but was restricted in certain distinct cerebellar areas only, while no differences in the distribution of the two hsps were found. The strongest response was detected in the Purkinje neurons but deep cerebellar nuclei were also positive. In no case glial cells were found to be reactive for hsps despite their strong response for specific markers like glial fibrillary acid protein (astrocytes) and cyclic nucleotide phosphodiesterase (oligodendrocytes). These data indicate that both the hsp70 and hsp90 family have fundamental physiological functions in cerebellar neurons while they seem to play only a minor role in the metabolism of glial cells.

The effect of etoposide (VP-16) on mouse L fibroblasts: modulation of stress response, growth and apoptosis genes.

Molecular events were studied in mouse L cells treated with etoposide (VP-16) a drug widely used in cancer therapy. Modulation of the expression of stress response genes belonging to the hsp70 family (hsp70, hsc73, grp78), growth- and cycle-related genes (c-myc, c-fos, c-jun, histone H3) and apoptosis-related genes (p53, TRPM-2, tTG) was monitored at different time points in the cells that remained adherent to the substrate up to 96 hours after exposure to VP-16. The steady state level of mRNA was determined by Northern blot analysis and hybridization with specific probes, and the relative rate of gene transcription was monitored by run on transcription with isolated nuclei. Our results indicate that protracted VP-16 treatment of 1. cells induces, within 24 hours, the arrest of DNA synthesis, repression of growth-related genes and transient induction of the tTG gene. Altogether these molecular events may contribute to the cytotoxic effect of VP-16. However in cells surviving a longer exposure to the drug, the expression of growth-related genes resumes, even if a blockade in DNA replication persists, and expression of the grp78 gene significantly increases. These data suggest that under continuous treatment with VP-16 a fraction of L cells showing increased resistance to the drug may emerge.

Detection and subcellular localization of an AML1 chimeric protein in the t(8;21) positive acute myeloid leukemia.

AML1, a gene encoding a protein of the PEBP2/CBF family of transcription factors is disrupted by translocations associated with human leukemia. In the t(8;21) acute myelogenous leukemia (AML), AML1 was found fused to a gene on chromosome 8 that we designated CDR (also known as ETO and MTG8). Immunoprecipitation experiments followed by immunoblotting using a combination of antibodies against different epitopes of one of the predicted chimeric proteins encoded by a fully characterized fusion transcript enabled us to visualize a chimeric protein in the t(8;21) Kasumi-1 cell line. The estimated size of this protein is 64 kDa. Immunoblotting of leukemic blasts containing the t(8;21) detected a protein of the same size. Immunofluorescence experiments indicate that the chimeric protein is localized in the nucleus. A normal AML1 protein of 27 kDa was also detected in t(8;21) Kasumi-1 cells. It remains to be established by which mechanism the mutant AML1 isoform may contribute to the leukemogenesis process of t(8;21)-positive acute myeloid leukemia.

Heat shock response in the liver: expression and regulation of the hsp70 gene family and early response genes after in vivo hyperthermia.

Heat shock response in cultured cells has been studied extensively; however few data are available on heat shock response in an intact organ of a living animal. In this study we analyzed the kinetics of expression of the heat shock protein 70 gene family (heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78) in the liver of the thermally stressed rat. New synthesis of heat shock protein 70 and heat shock cognate protein 73 was shown in liver slices pulse labeled in vitro with 35S-methionine. Accumulation of heat shock protein 70 and heat shock cognate protein 73 proteins was shown in total cellular extracts. 32P-labeled complementary DNA probes encoding heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78 were used to show that the levels of the corresponding messenger RNAs increase as a fraction of total RNA and in polysomes at different extents and with different kinetics. The induction of heat shock protein 70 and heat shock cognate protein 73 messenger RNAs reflected the increase in the synthesis of the corresponding proteins. Run-on transcription analysis indicated that the expression of heat shock protein 70 and heat shock cognate protein 73 genes was mainly regulated at the transcriptional level. On the contrary, both transcriptional and posttranscriptional regulatory mechanisms can explain the induction of the glucose-regulated protein 78 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein synthesis and gene expression in transplanted and postischemic livers.

The expression of some genes has been comparatively studied in transplanted rat liver and in liver reperfused after ischemia in situ. Experiments on protein synthesis by tissue slices from cold-stored or transplanted livers show that rat livers that retain a good capacity for protein synthesis during storage undergo a profound impairment in the capacity for protein synthesis during the first hours after transplantation. This recovers in the following hours. There is never any indication of synthesis of stress proteins, and of hsp 70 in particular. The steady-state level of mRNAs for albumin, transferrin, and beta-actin, which are well expressed in reperfused postischemic livers in vivo, are reduced early after transplantation and recover only many hours later. Run-on analysis shows that an early defect in transcription and a partial recovery of this process later on are responsible for these changes. The steady-state levels of the same mRNAs are well maintained in donor livers preserved in University of Wisconsin solution for at least 12 hr, and less satisfactorily in Euro-Collins solution. Results of run-on analysis parallel the data on mRNA levels. The behavior of these mRNAs is, therefore, clearly different in reperfused and transplanted liver. The early stages of liver transplantation seem to be characterized by a depressed capacity of gene expression, without the reactive phenomenon of activation of stress protein genes that occurs in reperfused postischemic livers.

Expression of HSP 70, immediate-early response and heme oxygenase genes in ischemic-reperfused rat liver.

BACKGROUND: Reperfusion of the liver after non-necrogenic ischemia induces the expression of the HSP gene and the synthesis of the hsp 70 protein, the best known among stress (heat-shock) proteins. EXPERIMENTAL DESIGN: We have studied the time course of the induction and the effects of cycloheximide treatment on the expression of c-fos, c-jun and the heat-shock gene HSP 70 in ischemic-reperfused livers; extracts of these livers have also been examined for the binding to a synthetic oligonucleotide containing the heat-shock consensus sequence (HSE) in order to reveal the possible presence of an active heat-shock factor (HSF) in ischemic-reperfused tissue. RESULTS: Expression of HSP 70 gene appears only after a certain threshold of cell damage, is preceded by induction of c-fos and c-jun but does not depend on ongoing protein synthesis. The binding of HSF to HSE seems to start during the late period of ischemia, although the subsequent reperfusion increases the effect. The level of heme-oxygenase mRNA, an indicator of oxidative stress, increases in the liver after reperfusion but the oxidative stress caused by CoCl2 treatment does not induce the expression of HSP 70 gene under the conditions of the present experiments. CONCLUSIONS: We suggest that, similar to heat-shock, protein malfolding occurring during ischemia may trigger the HSP 70 gene induction, which is then amplified by the subsequent reperfusion stress. A model of chemically induced oxidative stress seems to be unable to induce the HSP 70 gene expression with the same characteristics of heat shock or ischemia-reperfusion.

Stress proteins and reperfusion stress in the liver.

Blood reperfusion after temporary liver ischemia induces the expression of heat shock genes and the synthesis of heat shock proteins (hsps), in particular hsp 70. Induction requires a certain duration of ischemia, suggesting that cell damage before reperfusion is essential for activation of heat shock genes. The expression of the hsp 70 gene is preceded by activation of the cellular protooncogenes c-fos and c-jun. However, the product of these genes, which is transcription factor AP-1, seems unnecessary for activation of the hsp 70 gene, which does not require the integrity of protein synthesis. Hsp genes seem to behave as "early response genes," enabling the cell to respond to emergency situations.

Ferritin synthesis on polyribosomes attached to the endoplasmic reticulum.

The evidence that ferritin is synthesized both on free polyribosomes and on polyribosomes attached to the endoplasmic reticulum is reviewed. Evidence that some ferritin is secreted from cells after synthesis on bound polyribosomes was found to be inconclusive.

Ferritin mRNAs on rat liver membrane-bound polysomes synthesize ferritin that does not translocate across membranes.

Ferritin is a typical intracellular protein but small amounts are also present in serum and other biological fluids. The source and physiological significance of serum ferritin are still obscure. The presence of ferritin mRNAs on polysomes bound to endoplasmic reticulum (ER) could be relevant for the secretion of ferritin. By Northern blot analysis we found significant amounts of both L and H subunit mRNAs on rat liver membrane-bound polysomes. Immunoprecipitation of translational products of membrane-bound polysomes with anti-rat liver ferritin antibody showed that ferritin is actually synthesized on ER membranes. Analysis of RNA extracted from salt-washed rat liver microsomes demonstrated that ferritin mRNAs are translated by polysomes tightly bound to ER membranes. Following iron treatment, both the amount of H and L subunit mRNAs and ferritin synthesis increased sharply in both free and bound polysomal fractions. Translation of membrane-bound polysomes in the presence of microsomal membranes indicated that ferritin is not processed by signal sequence cleavage or glycosylation and is not translocated into ER membranes. Ferritin mRNAs found on membrane-bound polysomes are associated with ER in a specific way, however, their products do not seem to follow the classic secretory pathway and therefore the significance of the large amount of ferritin mRNAs in the bound ribosome fraction remains unclear.

Expression of the HSP 70 gene family in rat hepatoma cell lines of different growth rates.

We have studied the expression of different members of the HSP 70 gene family in MH1C1, FAO, and 3924A hepatoma cell lines, which possess different growth rates and show different levels of histone H3 gene expression. The cells have been subjected to mild (42 degrees C/1 h) or severe (45 degrees C/25 min) heat shock that causes a decrease in cell proliferation and histone H3 gene expression correlated to the severity of stress: previous mild heat shock protects against the effects of the subsequent severe exposure. All cell lines, irrespective of their growth rate, show a high constitutive expression of the HSC 73 gene, which is barely detectable in normal liver, and a good induction of the heat-inducible HSP 70 gene, which, however, seems to be induced less than in the normal tissue. The relative amount of grp 78 mRNA is high in all hepatoma cells lines, but only FAO cells maintain a significant expression of the albumin gene. The basic diversity in HSP 70 family gene expression between normal and tumors is still maintained in hepatoma cell lines, but the growth-related, quantitative differences among the transplantable hepatomas that we previously found in the animal (Bardella et al., Br. J. Cancer 55, 642-645, 1987; Cairo et al., Hepatology 9, 740-746, 1989), seem to be lost, or at least strongly blunted, in vitro.

Expression of the genes for the ferritin H and L subunits in rat liver and heart. Evidence for tissue-specific regulations at pre- and post-translational levels.

The proportion of ferritin light-chain and heavy-chain subunits (L and H) present in the ferritin multimeric shell varies between different tissues. To identify the regulatory mechanisms responsible for the greater amount of L in liver than in heart isoferritins, we analysed ferritin-gene expression at the RNA and protein levels in these two tissues of the rat. In the heart the ratio between the amount of L and H, at the level both of synthesis and accumulation, is about 1 and is the same as the ratio between their respective mRNAs. In contrast, in the liver, the ratio between the L- and H-mRNAs is approx. 2 and cannot entirely explain the large predominance of L in isoferritins in this tissue. Since in the liver the L-mRNA is neither preferentially associated with polyribosomes nor translated more efficiently than its H- counterpart, it seems that the liver-specific isoferritin profile is determined by a combination of pre- and post-translational mechanisms, whereas in heart the post-translational regulation does not seem to be relevant and the tissue-specific pattern is determined at the level of mRNA accumulation.

Liver gene expression during chronic dietary iron overload in rats.

To clarify the pathogenesis of hepatic iron toxicity, we investigated the effect of chronic dietary iron overload on the expression of several genes in rat liver. After 10 wk of iron treatment, when only minor histological features of liver damage were appreciable, the level of pro-alpha 2(I)-collagen mRNA was already higher than in control liver and increased further at 30 wk of treatment. Also, the relative amount of L ferritin subunit mRNA was enhanced early by iron load and was even more elevated at the latest time point considered, whereas neither H ferritin subunit nor transferrin mRNA levels were affected by iron treatment. In contrast, after chronic iron treatment, no variations were found in the steady-state level of mRNAs transcribed from liver-specific and preferentially expressed genes (albumin, alpha-fetoprotein, apolipoprotein A-1), growth-related genes (c-myc, c-Ha-ras and c-fos) and stress-induced genes (heat shock protein 70). These results suggest that chronic dietary iron overload in rats can specifically activate target genes in the liver (i.e., L ferritin and procollagen) in the absence of either histological signs of severe liver damage or alterations in differentiated liver functions.

Reprogramming of gene expression in postischemic rat liver: induction of proto-oncogenes and hsp 70 gene family.

Steady-state levels of messenger RNA (mRNA) for different members of the heat-shock protein 70 gene family were studied in rat livers reperfused after non-necrogenic ischemia. The expression of constitutive hsc 73 gene decreases during ischemia, returns to normal upon reperfusion, and increases 4 hr after restoration of blood flow. Reperfusion induces the expression of another hsp 70 gene family member (the so-called inducible hsp 70 gene), which remains at high levels for at least 7 hr. The induction of hsp 70 family genes is preceded by activation of the cellular oncogene c-fos, the most prompt change in gene expression detected in reperfused liver. Run-on experiments demonstrate that the increased expression of these genes is largely dependent on activation of transcription. Changes in the amount of c-myc and ornithine decarboxylase mRNA are not evident, while the level of the mRNA for glucose-regulated protein GRP 78 increases later, concurrent with the onset of the acute phase response to surgical trauma. Analysis of polysomal and nonpolysomal fractions from sucrose gradients indicates that in postischemic liver, hsp 70 and hsc 73 mRNA are rapidly engaged on light polysomal or nonpolysomal complexes and are later shifted to polysomes. Albumin mRNA displays the same behavior, indicating that hsp 70 mRNA are not preferentially translated and that increased transcription is the major mechanism for enhanced hsp synthesis in postischemic liver. Damage by active oxygen species, pressure overload, and derangements of protein synthesis is likely to include the causative factors of increased expression of c-fos and the hsp 70 gene family in postischemic reperfused liver.

Translational regulation of ferritin synthesis in rat liver. Effects of chronic dietary iron overload.

In rats with chronic dietary iron overload, a higher amount of liver ferritin L-subunit mRNA was found mainly engaged on polysomes, whereas in control rats ferritin L-subunit mRNA molecules were largely stored in ribonucleoprotein particles. On the other hand, ferritin H-subunit mRNA was unchanged by chronic iron load and remained in the inactive cytoplasmic pool. In agreement with previous reports, in rats acutely treated with parenteral iron, only the ferritin L-subunit mRNA increased in amount, whereas both ferritin subunit mRNAs shifted to polysomes. This may indicate that, whereas in acute iron overload the hepatocyte operates a translation shift of both ferritin mRNAs to confront rapidly the abrupt entry of iron into the cell, during chronic iron overload it responds to the slow iron influx by translating a greater amount of L-subunit mRNA to synthesize isoferritins more suitable for long-term iron storage.