Heat Shock-induced Cell Death Research Articles

Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.

Inhibition of BETs prevents heat shock-induced cell death via upregulating HSPs in SV40 large T antigen transfected cells.

Heat shock response is a protected mechanism against environmental changes for the organism, which must be tightly regulated. Bromodomain and extra terminal-containing protein family (BETs) regulate numerous gene expression in many physiological and pathological conditions, including viral infection. SV40 is considered as a highly human disease-associated virus. We aimed to explore whether BETs play a role in heat shock in SV40 large T antigen transfected cells. SV40LTA was transfected in HeLa cells using the Lipofectamine 8000. BETs inhibitor JQ1 and I-BET-762 was employed to treat transfected cells and HEK-293T cells. Heat shock treatment was performed to determine the effect of JQ1 and I-BET-762 on these cells. Western blot and quantitative RT-PCR were carried out to assess the expression of HSP70 and other HSPs. We found that inhibition of BETs by JQ1 and I-BET-762 protects cells from heat shock-induced death in HEK293T cells. Both JQ1 and I-BET-762 induce the expression of HSPs and HSF1 in HEK-293T cells. However, neither JQ1 nor I-BET-762 fail to induce the expression of HSPs in either HeLa or HBL-1 cells. When SV40 large T antigen was transfected into HeLa cells, the induction of HSP70 expressing and the protection of heat shock-induced cell death are reproduced by JQ1 and IBET treatment in these transfected cells. Inhibition of BETs by JQ1 and I-BET-762 prevents heat shock-induced cell death via upregulating HSPs in SV40 large T antigen transfected cells. Our data indicate a novel function of BETs in SV40 large T antigen transformed cells, affecting HSPs and HSF1 as well as its function on heat shock response.

Measuring and Perturbing Ferroptosis in Plants.

Ferroptosis is an oxidative iron-dependent cell death that was recently described in vertebrates, invertebrates, fungi, plants, and bacteria. In plants, ferroptosis has been reported in response to heat shock in roots of 6-day-old Arabidopsis thaliana seedlings. Generally, all biochemical and morphological ferroptosis hallmarks are conserved between animals and plants. Here, we describe a protocol to induce and quantify ferroptosis in plants based on the analysis of dead cells with a Sytox Green stain. Furthermore, heat shock induced cell death is prevented by using specific ferroptosis inhibitors.

Zinc supplementation prior to heat shock enhances HSP70 synthesis through HSF1 phosphorylation at serine 326 in human peripheral mononuclear cells

Acute alteration of the functional zinc status increases the HSP70 protein level after heat shock in peripheral blood mononuclear cells (PBMC), while zinc deficiency, but not zinc supplementation, further augments heat shock-induced cell death.

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death

BackgroundAnimals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant “apoptosis-like programmed cell death” (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies.ResultsHere, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40–55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40–55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents.ConclusionsWe conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

PHLDA1 Does Not Contribute Directly to Heat Shock-Induced Apoptosis of Spermatocytes.

Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat shock-induced cell death in spermatogenic cells, yet its precise physiological role is not well understood. Aiming to elucidate the hypothetical role of PHLDA1 in HSF1-mediated apoptosis of spermatogenic cells we characterized its expression in mouse testes during normal development and after heat shock. We stated that transcription of Phlda1 is upregulated by heat shock in many adult mouse organs including the testes. Analyzes of the Phlda1 expression during postnatal development indicate that it is expressed in pre-meiotic or somatic cells of the testis. It starts to be transcribed much earlier than spermatocytes are fully developed and its transcripts and protein products do not accumulate further in the later stages. Moreover, neither heat shock nor expression of constitutively active HSF1 results in the accumulation of PHLDA1 protein in meiotic and post-meiotic cells although both conditions induce massive apoptosis of spermatocytes. Furthermore, the overexpression of PHLDA1 in NIH3T3 cells leads to cell detachment, yet classical apoptosis is not observed. Therefore, our findings indicate that PHLDA1 cannot directly contribute to the heat-induced apoptosis of spermatocytes. Instead, PHLDA1 could hypothetically participate in death of spermatocytes indirectly via activation of changes in the somatic or pre-meiotic cells present in the testes.

Abstract 3301: Inhibition of the secretory pathway calcium ATPase 1 (SPCA1) in MDA-MB-231 breast cancer cells and the effect on protein expression

Abstract SPCA1 is a Ca2+ pump located on the membrane of the Golgi apparatus and is involved in controlling calcium levels within the Golgi. We have previously shown that SPCA1 is upregulated in some basal-like breast cancers. Given the role of the Golgi in protein trafficking and processing, we hypothesised that SPCA1 may be involved in regulating the expression of proteins important in cancer. To address this hypothesis we used 2D-DIGE to identify proteins that are altered when SPCA1 is silenced in MDA-MB-231 basal cancer cells. MDA-MB-231 cells were seeded into 6-well plates (75,000 cells/well) and treated with SPCA1 or non-targeting siRNA. Protein was isolated 72 h post siRNA treatment, and SPCA1 silencing was confirmed by immunoblotting. 2D-DIGE and MS/MS were used to identify proteins with differential expression with SPCA1 silencing. Heat shock protein 60 (HSP60) was identified as a protein potentially sensitive to SPCA1 silencing using 2D-DIGE. Immunoblotting and real time RT-PCR showed that SPCA1 silencing reduced HSP60 protein and mRNA expression by 81±2% and 60±7% (n=3, P<0.05), respectively. Given the role of HSP60 in cell death pathways, we also assessed the consequences of SPCA1 silencing on heat shock and staurosporine-induced cell death. SPCA1 silencing had no effect on heat shock-induced cell death but reduced the sensitivity of MDA-MB-231 breast cancer cells to staurosporine. Although further studies are required to fully characterize the functional consequences of HSP60 downregulation upon SPCA1 silencing, this work suggests that SPCA1 is a complex regulator of proteins important in cancer pathways. Citation Format: Jane M. Lee, Diana G. Ross, Gregory R. Monteith, Sarah J. Roberts-Thomson. Inhibition of the secretory pathway calcium ATPase 1 (SPCA1) in MDA-MB-231 breast cancer cells and the effect on protein expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3301. doi:10.1158/1538-7445.AM2014-3301

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax−/−Bak−/− cells and better than either Bid−/− or dominant-negative caspase-9-expressing cells. Only Bim−/− and Bax−/−Bak−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid−/− cells, it readily did so in Bim−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-XL with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.

Yeast Bax Inhibitor, Bxi1p, Is an ER-Localized Protein That Links the Unfolded Protein Response and Programmed Cell Death in Saccharomyces cerevisiae

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death.

Programmed Cell Death and Adaptation: Two Different Types of Abiotic Stress Response in a Unicellular Chlorophyte

Eukaryotic microalgae are highly suitable biological indicators of environmental changes because they are exposed to extreme seasonal fluctuations. The biochemical and molecular targets and regulators of key proteins involved in the stress response in microalgae have yet to be elucidated. This study presents morphological and biochemical evidence of programmed cell death (PCD) in a low temperature strain of Chlorella saccharophila induced by exposure to NaCl stress. Morphological characteristics of PCD, including cell shrinkage, detachment of the plasma membrane from the cell wall, nuclear condensation and DNA fragmentation, were observed. Additionally, a significant production of H(2)O(2) and increase in caspase 3-like activity were detected. We demonstrated that singly applied environmental stresses such as warming or salt stress trigger a pathway of PCD. Intriguingly, the prior application of salt stress seems to reduce heat shock-induced cell death significantly, suggesting a combined effect which activates a defense mechanism in algal cells. These results suggest that C. saccharophila can undergo PCD under stress conditions, and that this PCD shares several features with metazoan PCD. Moreover, the simultaneous exposure of this unicellular chlorophyte to different abiotic stresses results in a tolerance mechanism.

'Heated' debates in apoptosis.

Hippocrates' assertion that 'what the lance does not heal, fire will' underscores the fact that for thousands of years heat has been used to treat a variety of diseases, including cancer. Indeed, spontaneous tumor remission has been observed in patients following feverish infection [1], and expression of activated oncogenes, such as Ras, can render tumor cells sensitive to heat compared with normal cells [2, 3]. In the past, a primary drawback to the use of heat as a clinical therapy was the inability to selectively focus heat to tumors in situ. Of late, however, several approaches have been devised to deliver heat more precisely, including the use of heated nanoparticles, making hyperthermia a more clinically tractable treatment option [4, 5]. Despite these practical advances, the mechanisms responsible for heat shock-induced cell death remain controversial and ill-defined. In this Visions and Reflections we discuss recent findings surrounding the initiation of heat shock-induced apoptosis, and propose future areas of research.

Heat Stress: an Inducer of Programmed Cell Death in Chlorella saccharophila

Programmed cell death (PCD) has been recognized as a fundamental cellular process conserved in metazoans, plants and yeast. However, the cellular mechanisms leading to PCD have not been fully elucidated in unicellular organisms. Evidence is presented that heat stress induces PCD in Chlorella saccharophila cells. Our results demonstrate that heat shock triggers a PCD pathway occurring with characteristics features such as chromatin condensation, DNA fragmentation, cell shrinkage and detachment of the plasma membrane from the cell wall, and suggest the presence of caspase 3-like activity. The caspase 3 inhibitor Ac-DEVD-CHO gave significant protection against heat shock-induced cell death. Moreover, a reduction in photosynthetic pigment contents associated with alteration of chloroplast morphology and a fairly rapid disappearance of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and the light-harvesting complex of PSII have been observed. The timing of events in the signaling cascade associated with the C. saccharophila heat shock PCD response is discussed. Insights into this field may have general implications for understanding the pathway of cell death in unicellular green algae.

Heat shock does not induce γH2AX foci formation but protects cells from N-methyl- N′-nitro- N-nitrosoguanidine-induced genotoxicity

The involvement of DNA damage in heat shock-induced cell death remains controversial. To investigate whether heat shock can induce DNA damage, we tested the induction of γH2AX foci formation, a sensitive indicator for DNA double strand breaks (DSBs), by heat shock treatment in several cell lines including HeLa, CHL, HepG2, and 293 cells, as well as human spermatozoa. Although heat shock treatment can decrease cell viability, no induction of γH2AX foci formation was observed in any of these cells. In addition, a p53-deficient cell line (U2OSE6tet24) and a flap endonuclease 1 (FEN1)-deficient cell line (FL-FEN1 −) also did not show induction of γH2AX foci after heat shock treatment. Finally, it was found that 30 min of pre-heat shock can inhibit γH2AX foci formation induced by an alkylating agent, N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), which is known to induce γH2AX foci formation. On the other hand, heat shock after MNNG treatment did not affect the γH2AX foci formation induced by MNNG. Taken together, these data suggest that although heat shock might influence the γH2AX foci formation process, it does not induce DNA damage in the cells tested in this study.

A novel HSF1-mediated death pathway that is suppressed by heat shock proteins

Heat shock response is an adoptive response to proteotoxic stress, and a major heat shock transcription factor 1 (HSF1) has been believed to protect cells from cell death by inducing heat shock proteins (Hsps) that assist protein folding and prevent protein denaturation. However, it is revealed recently that HSF1 also promotes cell death of male germ cells. Here, we found a proapoptotic Tdag51 (T-cell death associated gene 51) gene as a direct target gene of HSF1. Heat shock and other stresses induced different levels of Hsps and Tdag51, which depend on cell types. Hsps bound directly to the N-terminal pleckstrin-homology like (PHL) domain of Tdag51, and suppressed death activity of the C-terminal proline/glutamine/histidine-rich domain. Tdag51, but not major Hsps, were induced in male germ cells exposed to high temperatures. Analysis of Tdag51-null testes showed that Tdag51 played substantial roles in promoting heat shock-induced cell death in vivo. These data suggest that cell fate on proteotoxic condition is determined at least by balance between Hsp and Tdag51 levels, which are differently regulated by HSF1.

Cytochrome c Is Released in a Reactive Oxygen Species-Dependent Manner and Is Degraded via Caspase-Like Proteases in Tobacco Bright-Yellow 2 Cells en Route to Heat Shock-Induced Cell Death

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.

Arabidopsis Bax inhibitor‐1 functions as an attenuator of biotic and abiotic types of cell death

Programmed cell death (PCD) is a common process in eukaryotes during development and in response to pathogens and stress signals. Bax inihibitor-1 (BI-1) is proposed to be a cell death suppressor that is conserved in both animals and plants, but the physiological importance of BI-1 and the impact of its loss of function in plants are still unclear. In this study, we identified and characterized two independent Arabidopsis mutants with a T-DNA insertion in the AtBI1 gene. The phenotype of atbi1-1 and atbi1-2, with a C-terminal missense mutation and a gene knockout, respectively, was indistinguishable from wild-type plants under normal growth conditions. However, these two mutants exhibit accelerated progression of cell death upon infiltration of leaf tissues with a PCD-inducing fungal toxin fumonisin B1 (FB1) and increased sensitivity to heat shock-induced cell death. Under these conditions, expression of AtBI1 mRNA was up-regulated in wild-type leaves prior to the activation of cell death, suggesting that increase of AtBI1 expression is important for basal suppression of cell death progression. Over-expression of AtBI1 transgene in the two homozygous mutant backgrounds rescued the accelerated cell death phenotypes. Together, our results provide direct genetic evidence for a role of BI-1 as an attenuator for cell death progression triggered by both biotic and abiotic types of cell death signals in Arabidopsis.

N-t-Butyl hydroxylamine regulates heat shock-induced apoptosis in U937 cells

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Therefore, compounds that scavenge reactive oxygen species may regulate heat shock-induced cell death. Recently, it has been shown that the decomposition product of the spin-trapping agent α-phenyl-N-t-butylnitrone, N-t-butyl hydroxylamine (NtBHA), mimics α-phenyl-N-t-butylnitrone and is much more potent in delaying reactive oxygen species-associated senescence. We investigated the protective role of NtBHA against heat shock-induced apoptosis in U937 cells. Upon exposure to heat shock, there was a distinct difference between the untreated cells and the cells pre-treated with 0.1 mM NtBHA for 2 h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. Upon exposure to heat shock, NtBHA pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to untreated cells. This study indicates that NtBHA may play an important role in regulating the apoptosis induced by heat shock, presumably through scavenging of reactive oxygen species.

A Novel Arabidopsis Gene Causes Bax-like Lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.

Human Fas-associated Factor 1 Interacts with Heat Shock Protein 70 and Negatively Regulates Chaperone Activity

We examined the cell death-inducing property of human Fas-associated factor 1 (hFAF1) in the heat shock signaling pathway. By employing co-immunoprecipitation and peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that hFAF1 binds to the 70-kDa heat shock protein family (Hsc70/Hsp70). Interaction mapping indicated that the 82-180 sequence of hFAF1 directly binds to the N-terminal region containing sequence 1-120 of Hsc70/Hsp70. This binding is very tight regardless of ATP and heat shock treatment. Hsc70/Hsp70 and hFAF1 co-localized in the cytosol and nucleus and concentrated to the perinuclear region by heat shock treatment. We examined how hFAF1 regulates Hsp70 function, and found that hFAF1 inhibited the Hsp70 chaperone activity of refolding denatured protein substrates, accelerated heat shock-induced SAPK/JNK activation, and raised heat shock-induced cell death in a binding dependent manner. These results suggest that hFAF1 prevents cells from recovery after stress by binding to and inhibiting the chaperone activity of Hsp70.

Screening of stress enhancer based on analysis of gene expression profiles: enhancement of hyperthermia-induced tumor necrosis by an MMP-3 inhibitor.

To improve the therapeutic benefit of hyperthermia, we examined changes of global gene expression after heat shock using DNA microarrays consisting of 12 814 clones. HeLa cells were treated for 1 h at 44 degrees C and RNA was extracted from the cells 0, 3, 6, and 12 h after heat shock. The 664 genes that were up or down-regulated after heat shock were classified into 7 clusters using fuzzy adaptive resonance theory (fuzzy ART). There were 41 genes in two clusters that were induced in the early phase after heat shock. In addition to shock response genes, such as hsp70 and hsp40, the stress response genes c-jun, c-fos and egr-1 were expressed in the early phase after heat shock. We also found that expression of matrix metalloproteinase 3 (MMP-3) was enhanced during the early response. We therefore investigated the role of MMP-3 in the heat shock response by examining HeLa cell survival after heat treatment in the presence and absence of an MMP-3 inhibitor, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycylhydroxamic acid (NNGH) or N-hydroxy-2(R)-[[4- methoxysulfonyl](3-picolyl)amino]-3-methylbutaneamide hydrochloride (MMI270). The number of surviving cells 3 days after heat treatment significantly decreased, reaching 3.5% for NNGH and 0.2% for MMI270. These results indicate that the MMP-3 inhibitors enhanced heat shock-induced cell death and behaved as stress enhancers in cancer cells. This valuable conclusion was reached as a direct result of the gene expression profiling that was performed in these studies.