Translocation of cytoplasmic HSP70 onto the surface of EL-4 cells during apoptosis

Heat shock proteins (HSPs) are involved in a variety of intracellular processes and can have both pro- and anti-apoptotic action. However, little is known about the role of HSPs in the progression of apoptosis. Translocation of HSPs to the surface of apoptotic cells is a previously observed phenomenon demonstrating participation of these proteins in execution of the terminal stages of apoptosis. In a previous study we showed that development of EL-4 lymphoma cell apoptosis in vitro is accompanied by elevation of surface HSP expression. In this study we used this model to analyse the relationship of HSP70 expression and its translocation to the cell surface during apoptosis with some key intracellular events. Our data demonstrate a synchronization of surface and intracellular HSP70 expression with bcl-2 expression, intracellular reactive oxygen species (ROS) concentration and caspase-3 activity. A maximum level of surface and intracellular HSP70 expression was observed at an irreversible phase of EL-4 cell apoptosis after mitochondrial potential depolarization. In addition, an enhancement of the relative level of cytoplasmic HSP70 translocation to the cell surface was concomitant with EL-4 cell apoptosis. However, the size of surface and intracellular pools of HSP70, increasing for initial and intermediate stages of cell death, decreased at the terminal phase of apoptosis. Western blot analysis of HSP70 in conditioned supernatant obtained from EL-4 cell tissue showed that the observed decrease of HSP70 cell content might be due to surface HSP70 shedding into the intercellular space.     

The protective role of HSP90 against 3-hydroxykynurenine-induced neuronal apoptosis

3-hydroxykynurenine (3HK), an endogenous metabolite of tryptophan in the kynurenine pathway, is a potential neurotoxin in several neurodegenerative disorders. Stabilizing protein structure, heat shock proteins (HSPs) have diverse roles as molecular chaperones to mediate stress tolerance. In the present study, we investigated the possible protective role of HSPs against 3HK induced neuronal cell death. Here we report that 3HK induced in a dose- and time-dependent manner neuronal cell death in neuroblastoma SK-N-SH cells. The cell death showed characteristic apoptotic features such as cell shrinkage, plasma membrane blebbing, chromatin condensation, and nuclear condensation and fragmentation. Furthermore, SK-N-SN cells were protected from 3HK induced cytotoxicity by prior elevation of HSPs expression. Our results show that the protective effect was abolished by HSP90 anti-sense oligonucleotides while not by HSP27 and HSP70 anti-sense oligonucleotides. Also, our result shows that HSP90 effectively inhibits caspases activities leading to the apoptosis. These results suggest that 3HK induces apoptosis in neuroblastoma SK-N-SN cells and that HSP90 is major contributing protein component of protection against 3HK induced apoptosis.     

Effect of chronic hypoxia on proliferation, apoptosis, and HSP70 expression in mouse bronchiolar epithelial cells

Heat shock proteins (HSPs) can be induced by various stresses and play an important role in cell cycle progression. HSP70 has been shown to act as an inhibitor of apoptosis. We studied HSP70 expression in bronchial epithelial cells of C57BL/6 mice and homozygous HPS70 knockout mice (hsp70.1-/-) exposed to chronic hypoxic stress. We also investigated changes in cellular proliferation and apoptosis in relation to HSP70. Lungs were removed from mice after a three-week period of exposure to 10 % O(2). Immunoblots for HSP70 and immunohistochemical staining for HSP70 and Ki-67 were performed. Apoptosis was assessed using the TUNEL assay. The three-week period of hypoxic stress did not change HSP70 levels in total lung tissue, but a significant reduction in HSP70 expression was observed in bronchiolar epithelial cells. In wild type mice, both HSP70 and Ki-67 expression were significantly reduced in bronchiolar epithelial cells. In homozygous HPS70 knockout mice (hsp70.1-/-), apoptosis of bronchiolar epithelial cells was significantly increased. Our results suggest that HSP70 may exert anti-apoptotic effects in mouse bronchiolar epithelial cells.     

Role of G1 phase in the UV-induced apoptosis of EL-4 mouse lymphoma cells

Although UV is known to induce apoptotic cell death to various animal cells, relationship between cell cycle and UV-induced apoptosis is still unclear. In this study, we investigated the role of G1 phase in UV-induced apoptosis by using EL-4 mouse lymphoma cells which have wild type p53. After 500 J/m UV irradiation, an increase of apoptotic fraction was accompanied by cell cycle accumulation in the G1 phase. Apoptotic fraction after UV-exposure was remarkably augmented by treatment with 2-AP, a G1 checkpoint inhibitor. In contrast, aphidicolin, an inhibitor of DNA polymerase , suppressed the rate of apoptotic fraction.These results suggest that mandatory cell cycle progression from G1 to S leaves the damaged DNA unrepaired and may increase the apoptotic fraction. To investigate the precise mechanism in the G1 phase, UV was exposed to the G1-synchronized cells and apoptotic fraction was serially observed. Synchronized EL-4 cells passed through the G1 phase in 8 h. Within the G1 phase, late-G1 cells (6 h after M) were more sensitive to UV-induced apoptosis than early-G1 cells (2 h after M) (49.7 ± 9.0% vs. 41.5 ± 8.5%, p < 0.05). In HL-60 cells, lacking in p53 expression, such a difference was not observed. Western blot analysis revealed that expression of p53 in synchronized EL-4 cells was increasingly enhanced during G1 phase. After UV-exposure, p53 expression gradually decreased in early-G1 cells, but it was kept at almost the same level in late-G1 cells. In addition, bcl-2 expression in early-G1 cells showed a more rapid and larger increase than that in late-G1 cells. These results suggest that susceptibility of the G1 cells to UV-induced apoptosis depends on their position within the G1 phase, and late-G1 is more sensitive than early-G1. Sensitivity to UV-induced apoptosis is closely related to the expression level of p53 and bcl-2 proteins. Early-G1 cells may be able to take enough time to repair damaged DNA until they reach the G1 checkpoint compared to the late-G1 cells.     

Heat shock proteins: endogenous modulators of apoptotic cell death

The highly conserved heat shock proteins (HSPs) accumulate in cells exposed to heat and a variety of other stressful stimuli. HSPs, which function mainly as molecular chaperones, allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked and HSPs induced in response to stress appear to function at key regulatory points in the control of apoptosis. HSPs include antiapoptotic and proapoptotic proteins that interact with a variety of cellular proteins. Their expression level can determine the fate of the cell in response to a death stimulus, and apoptosis-inhibitory HSPs, in particular HSP27 and HSP70, may participate in carcinogenesis. This review summarizes apoptosis-regulatory function of HSPs.     

Caspases participation in cell death induced by the GD2-specific antibodies

Contribution of the main caspases in cytotoxic effects induced by the monoclonal antibody 14G2a specific to the tumor-associated ganglioside GD2 was studied in the EL-4 mouse lymphoma cells. The constitutive expression of the procaspase genes was found in the EL-4 cells. Incubation of the cells with the 14G2a antibodies did not result in increasing of the procaspase synthesis. We also demonstrated with the use of fluorescently labeled substrates of caspases that the procaspase enzymatic activity was not enhanced. At an equal level of cell death, activities of caspase-3 and caspase-9 in the cells which were incubated with the 14G2a antibodies were 7.5 and 3 times lower, respectively, than those in the staurosporine-treated cells. The pan-caspase inhibitor (Z-VAD-FMK) and the caspase-3 inhibitor decreased the cytotoxic effects induced by the 14G2a antibodies by 9–16 and 6–13%, respectively. The staurosporine-induced level of the apoptosis decreased by 55–65% under the same conditions. Inhibitors of the initiation caspase-8 and caspase-9 had no influence on the antibody-induced cell death. The inhibitory analysis also demonstrated that the caspases were not involved in the triggering of the initial stages of the antibody-induced cell death such as apoptotic volume decrease and permeabilization of the cell plasma membrane. Thus, caspases did not play a key role in the cell death induced by the anti-GD2 antibodies, and their slight enzymatic activity did not determine the main mechanism of cell death mediated through the tumor-associated ganglioside GD2.     

Apoptosis Versus Cell Differentiation: Role of Heat Shock Proteins HSP90, HSP70 and HSP27

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs     

Apoptosis Versus Cell Differentiation

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.     

Surface expression of Bcl-2 in chronic lymphocytic leukemia and other B-cell leukemias and lymphomas without a breakpoint t(14;18)

Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

Evidence for a role of heat-shock proteins in proliferation after heat treatment of synchronized mouse neuroblastoma cells

A role for heat-shock proteins (HSPs) in proliferation after heat treatment was considered in synchronized mouse neuroblastoma cells. For this purpose enhancement of HSP synthesis after heat treatment was inhibited by actinomycin D and the effect of this on cell cycle progression into mitosis and on cell survival was studied both in thermoresistant G1- and in thermosensitive late S/G2-phase cells. In G1-phase cells expression of basal and heat-induced HSP synthesis was the same as that in late S/G2-phase cells, which suggests that regulation of thermoresistance throughout the cell cycle is not directly linked with HSP synthesis. The synthesis of HSP36, HSP68, and HSP70 was enhanced after a 30-min treatment at 41-43 degrees C. Increase of HSP synthesis after heat shock was partly suppressed by the presence of 0.1 microgram/ml actinomycin D during heat treatment, while 0.2 micrograms/ml prevented enhancement of HSP synthesis completely. Suppression of heat-induced HSP synthesis by actinomycin D had the same concentration dependency in G1- and late S/G2-phase cells. Actinomycin D potentiated induction of mitotic delay by heat treatment (30 min, 42.5 degrees C) but only under conditions where it actually inhibited heat-induced enhancement of HSP synthesis. Heat-induced cell killing was also potentiated by actinomycin D. The potentiating effect of actinomycin D on heat-induced mitotic delay and on heat-induced cell killing was more pronounced in G1-phase cells than in late S/G2-phase cells. These results give evidence for a role of HSPs in the resumption of proliferation after heat treatment and suggest that heated G1-phase cells are more dependent on HSP synthesis for recovery of proliferation after heat treatment than heated late S/G2-phase cells.     

Cytochemical identification of HSP110 during early mouse facial development.

Apoptotic cell death constitutes a common phenomenon observed during development. This process plays an important role in the regulation of cell populations and in early differentiation of embryonic organs. Several teratologic situations are considered as resulting in a dramatic increase of the apoptotic process. In mammalian cells, heat shock proteins (HSPs), expressed or increased in response to various stresses, act as molecular chaperones in physiological conditions. In order to determine specific histochemical markers of apoptotic cells in normal craniofacial development, we observed the expression of stress proteins (HSPs) 70, 86, and 110. The apoptotic pattern of mesectodermal cell death areas was confirmed using both nuclear staining (Feulgen) and specific labeling of DNA fragmentation (TUNEL). These areas are localized in the proximal parts of the first and second visceral arches. They are located in mesectodermal and ganglionic cells. Apoptotic mesectodermal populations strongly express HSP110, as shown by the cytochemical identification of HSP110 and by double staining HSP110-TUNEL, suggesting that this protein could be considered as a new marker for apoptotic embryonic cells, and could be used in further teratologic studies to better quantify induced cell death.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope.Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

几种热激蛋白在细胞凋亡信号通路中的调控作用

热激蛋白(heat shock proteins, HSPs)作为进化保守的蛋白家族 之一,普遍存在于各种生物体中,并在生物体内发挥着重要的生理功能.大 量的实验证据表明,热激蛋白与细胞凋亡密切相关,参与细胞凋亡信号通 路的多个环节. 近年来有关该领域的研究已获得了重要的突破与进展.一方 面,热激蛋白主要起着抑制细胞凋亡、促进细胞存活的作用;另一方面, 某些热激蛋白又能够作为凋亡蛋白的分子伴侣,促进细胞凋亡,比如HSP70 能够激活DNase来促使细胞凋亡,线粒体内HSP60能够促进caspase依赖的细 胞凋亡途径.本文在阐明细胞凋亡信号通路的基础上,综述了近年来几种不 同热激蛋白家族（HSP90、 HSP70 、HSP60和小分子HSPs）在细胞凋亡调控 中作用的研究进展,重点阐述了几种主要热激蛋白与细胞凋亡信号通路上 相关因子的相互作用,并绘制了热激蛋白在细胞凋亡信号通路中的调控图 ,为进一步完善细胞凋亡调控网络研究提供一定的参考.     

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.     

Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.     

Expression level of c-FLIP versus Fas determines susceptibility to Fas ligand-induced cell death in murine thymoma EL-4 cells

The caspase-8 inhibitor c-FLIP blocks death receptor-mediated cell death and plays an essential role in the regulation of lymphocyte homeostasis and the immune escape of tumors. The murine thymoma cell line EL-4 was resistant to Fas ligand (FasL)-induced apoptosis by constitutive expression of FLIP (L). Cycloheximide downregulated the expression of FLIP (L) and markedly sensitized EL-4 cells to FasL-induced apoptosis. In contrast, DNA-damaging agents sensitized EL-4 cells to FasL-induced cell death via an increase of cell-surface Fas without any influence on FLIP (L) expression. Enforced expression of transfected Fas rendered EL-4 cells highly susceptible to FasL-induced cell death. These findings demonstrate that susceptibility to FasL-induced cell death mainly depends on the expression level of c-FLIP versus cell-surface Fas.     

Attenuating effects of heat shock against TGF-beta1-induced apoptosis in cultured rat hepatocytes

Heat shock proteins (HSPs) induction confers protection against diverse forms of cellular injury. However, the mechanism by which HSPs exert cytoprotective effects remains unclear. Treatment of rat hepatocyte with transforming growth factor-beta1 (TGF-beta1) induces growth arrest followed by extensive cell death by apoptosis. In this study, the effects of preexposure to heat on TGF-beta1-induced apoptosis of cultured hepatocytes were examined. Treatment of hepatocytes for 24 h with TGF-beta1 resulted in significant apoptotic cell death, as demonstrated by DNA fragmentation, caspase activation, and hypodiploid DNA peak. Moreover, TGF-beta1-induced cell death was accompanied by an enhanced generation of reactive oxygen species and a loss of the mitochondrial membrane potential. These effects were attenuated when the hepatocytes were subjected to 43 degrees C for 20 min prior to the cytokine stimulation. The enhancement in HSP70 expression at mRNA and protein levels induced by heat preexposure was accompanied by an increase in mRNA levels of intracellular antioxidant enzymes. Heat treatment also prevented TGF-beta1-induced activation of nuclear factor kappa B (NF-kappaB) by preventing the degradation of the inhibitory protein kappa Balpha (IkappaBalpha). In conclusion, these data indicate that in the mechanism by which a mild heat pretreatment increases the resistance of hepatocytes to TGF-beta1-induced apoptotic cell death, the upregulation of catalase expression and a decrease in ROS generation are involved.     

Correlation of HSP110 expression with all-trans retinoic acid-induced apoptosis

In a previous study, we observed the strong expression of a stress protein of the HSP100/Clp family (HSP110) in apoptotic mesectodermal cells during early mouse facial development. In the present study, we describe the strong expression of the same HSP110 in mesectodermal cells undergoing apoptosis after all-trans retinoic acid (RA) administration. We used a teratological model known to increase cell deaths mainly in the first and second branchial arches during mammalian cephalogenesis: the treatment of E9 mouse embryos with all-trans RA, which results in craniofacial malformations comparable to those that characterize mandibulofacial dysostosis in man. Pregnant NMRI mice were treated with 60 mg/kg body weight of all-trans RA, given orally on day 9 of gestation; embryos were taken 4, 12 or 24 hr after RA administration. The apoptotic pattern of RA-induced cell deaths was confirmed using the dUTP biotin nick-end labeling (TUNEL) method and transmission electron microscopy (TEM). HSP110 expression was detected using an immunohistochemical approach. The increase in the number of TUNEL-positive cells and HSP110-positive cells after all-trans RA administration was quantified in the first branchial arch using a computerized method. Twelve hours after RA administration, the increase in the number of HSP110-positive cells is greater than the increase in the number of TUNEL-positive cells. Twenty-four hours after RA administration, only TUNEL-positive cells remain strong in number. We suggest that HSP110 expression could represent a biochemical event of apoptotic cell death induced by RA, associated with early stages of the apoptotic process. In order to find out if HSP110 expression resulted from neosynthesis, we performed in situ hybridization, which demonstrated that the expression of HSP110 occurred at the level of mRNA.