Apoptosis Stimuli Research Articles

Akt Attenuation of the Serine Protease Activity of HtrA2/Omi through Phosphorylation of Serine 212

The serine protease HtrA2/Omi is released from the mitochondria into the cytosol following apoptosis stimuli, leading to the programmed cell death in caspase-dependent and -independent manners. The function of HtrA2/Omi closely relates to its protease activity, which is required for cleavage of its substrate such as the members of the X-linked inhibitor of apoptotic protein family. However, the regulation of HtrA2/Omi by signaling molecule has not been documented. Here we report that serine/threonine kinases Akt1 and Akt2 phosphorylate mitochondria-released HtrA2/Omi on serine 212 in vivo and in vitro, which results in attenuation of its serine protease activity and pro-apoptotic function. Abolishing HtrA2/Omi phosphorylation by Akt through mutation of serine 212 to alanine (HtrA2/Omi-S212A) retains its serine protease activity and induces more apoptosis as compared with wild-type HtrA2/Omi. Conversely, HtrA2/Omi-S212D, a mutant mimicking phosphorylation, lost the protease activity and failed to induce the programmed cell death. Furthermore, the phosphorylated HtrA2/Omi fails to cleave X-linked inhibitor of apoptotic protein without interfering with their complex formation. In addition, Akt inhibits the release of HtrA2/Omi from the mitochondria into the cytoplasm in response to cisplatin treatment. These data reveal for the first time that HtrA2/Omi is directly regulated by Akt and provide a mechanism by which Akt induces cell survival at post-mitochondrial level.

Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family

To establish a vacuole that supports bacterial replication, Legionella pneumophila translocates a large number of bacterial proteins into host cells via the Dot/Icm type IV secretion system. Functions of most of these translocated proteins are unknown, but recent investigations suggest their roles in modulating diverse host processes such as vesicle trafficking, autophagy, ubiquitination, and apoptosis. Cells infected by L. pneumophila exhibited resistance to apoptotic stimuli, but the bacterial protein directly involved in this process remained elusive. We show here that SidF, one substrate of the Dot/Icm transporter, is involved in the inhibition of infected cells from undergoing apoptosis to allow maximal bacterial multiplication. Permissive macrophages harboring a replicating sidF mutant are more apoptotic and more sensitive to staurosporine-induced cell death. Furthermore, cells expressing SidF are resistant to apoptosis stimuli. SidF contributes to apoptosis resistance in L. pneumophila-infected cells by specifically interacting with and neutralizing the effects of BNIP3 and Bcl-rambo, two proapoptotic members of Bcl2 protein family. Thus, inhibiting the functions of host pro-death proteins by translocated effectors constitutes a mechanism for L. pneumophila to protect host cells from apoptosis.

Two Waves of Mitochondrion Disruption in Apoptosis: Implications for the Design of Anti-Cancer Drugs.

Apoptosis plays an important role in preventing cancer development and promoting anti-cancer therapy. During mitochondria-mediated apoptosis, the disruption of mitochondria leads to the release of cytochrome c to form apoptosome with Apaf-1 and caspase-9, resulting in caspase-9 activation. However, the precise mechanisms controlling mitochondrion disruption remain elusive. Ablation of caspase-9 or other apoptosome components causes abnormal brain development and perinatal lethality in mice, making it difficult to assess the in vivo functions of caspase-9 signaling in many cell types and the role of caspase-9 in regulating mitochondria disruption. Through conditional knockout and inducible activation of caspase-9, we show that the loss of mitochondrion membrane potential (Δψm) during apoptosis proceeds in distinct caspase-9-dependent and independent steps. The initial loss of cells with high Δψm (Δψmh) was independent of caspase-9, but required BH3-only proteins of the Bcl-2 family, such as Bim and BNIP3L. Caspase-9 deficiency arrested the B cells at a stage with intermediate levels of Δψm (Δψmint). Apoptosis stimuli induced activation of effector caspases and cleavage of anti-apoptotic myeloid cell leukemia-1 (Mcl-1) in wild type but not caspase-9−/− B cells. Specific activation of caspase-9 by chemical-induced dimerization in human leukemia H9 cells also resulted in the loss of Δψm and cleavage of Mcl-1, but silencing of downstream effector caspases inhibited Mcl-1 cleavage and sequestered the cells at the Δψmint stage. Moreover, a cleavage-resistant Mcl-1 inhibited caspase-9-dependent mitochondrion disruption and apoptosis, indicating that caspase-9-mediated mitochondrion destruction involves Mcl-1 processing. Our data suggest that BH3-only proteins initiate the first wave of mitochondrion disruption during apoptosis with Δψmh to Δψmint transition, while caspase-9 signaling orchestrated the second wave of Δψmint to Δψm− transition through the cleavage of Mcl-1. Defects in mitochondria-mediated apoptosis pathway are associated with the development and treatment failures of many hematological malignancies. Identification of stepwise mitochondrial disruption may allow specific targeting of apoptotic machineries in various therapeutic settings. Chemical-induced dimerization of caspase-9 may be particularly useful for the design of anti-cancer drugs to treat those malignancies involving Mcl-1 over-expression.

Structure of the Calmodulin αII-Spectrin Complex Provides Insight into the Regulation of Cell Plasticity

AlphaII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of alphaII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of alphaII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, we have solved the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human alphaII-spectrin (Protein Data Bank ID code 2FOT). The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes.

Reduced semen quality in chronic prostatitis patients that induce the release of apoptotic protein Omi/HtrA2 from spermatozoa

The relationship between chronic prostatitis and fertility has been disputed for many years. Several groups have shown infection and autoimmune response against prostate antigens could have a deleterious effect on semen quality and fertility. This study was conducted to test the hypothesis that Omi/HtrA2-induced apoptosis in chronic prostatitis could be a mechanism underlying the observed clinical benefit. The Omi/HtrA2 serine protease is a nuclear-encoded mitochondrial protein, which can be released from mitochondria into the cytosol after apoptosis stimuli, inducing apoptosis in caspase-dependent and independent manners. Forty-one patients diagnosed as suffering from chronic prostatitis were included. Healthy normal individuals were included as controls. Human spermatozoa in the semen were purified by Percoll-gradient technique to separate the seminal plasma and other round cells. Measurements for sperm concentration, motility, morphology, proinflammatory cytokines, Omi/HtrA2 mRNA and protein levels in spermatozoa of chronic protatitis patients, were performed accordingly. Significantly increased levels of proinflammatory cytokines were detected in seminal plasma from these prostatitis patients. Omi/HtrA2 mRNA and protein levels were significantly higher in prostatitis men than in normal men. This study shows that chronic prostatitis patients present important alterations in their semen quality parameters, Omi/HtrA2 mRNA and protein levels of spermatozoa. We speculate that the inflammatory process involved may affect male fertility by release of proapoptotic protein Omi/HtrA2.

Endoplasmic reticulum stress and trophic factor withdrawal activate distinct signaling cascades that induce glycogen synthase kinase-3β and a caspase-9-dependent apoptosis in cerebellar granule neurons

Loss of trophic or activity-dependent survival signals is commonly recognized as a stimulus for neuronal apoptosis and may play a significant role in neurodegeneration. Recent data have also implicated endoplasmic reticulum (ER) stress as an important factor in some neurodegenerative conditions. However, whether shared or unique apoptotic cascades are activated by trophic factor withdrawal (TFW) versus ER stress in primary neurons has not previously been investigated. In primary cultures of rat cerebellar granule neurons (CGNs), the ER stressor brefeldin A activated a discrete pathway involving the following: (1) stimulation of the ER resident kinase PERK, (2) enhanced phosphorylation of the translation initiation factor eIF2α, and (3) increased expression and nuclear localization of the transcription factor Gadd153/CHOP. ER stress-induced CGN apoptosis was blocked by an antagonist of IP3 receptor-mediated Ca2+ release, 2-aminoethoxydiphenyl borate (2-APB), and by expression of ER-targeted Bcl-2. In contrast, CGN apoptosis elicited by TFW (i.e., removal of serum and depolarizing extracellular potassium) did not display any ER stress component nor was it blocked by either 2-APB or ER-Bcl-2. Despite these apparent differences, both brefeldin A and TFW induced dephosphorylation (activation) of glycogen synthase kinase-3β (GSK-3β). Moreover, inhibitors of GSK-3β (IGF-I, lithium) and caspase-9 (LEHD-fmk) significantly protected CGNs from apoptosis induced by either ER stress or TFW. These data indicate that ER stress and TFW elicit distinct signals that activate GSK-3β and intrinsic apoptosis in neurons.

Nuclear Localization of the Serine/Threonine Kinase DRAK2 Is Involved in UV-Induced Apoptosis

DAP kinase-related apoptosis-inducing kinase 2 (DRAK2), a member of the DAP kinase family, is a serine/threonine kinase capable of inducing apoptosis. Here we studied the relationship between DRAK2 intracellular localization and apoptosis, and found that UV light acts as a stimulus for apoptosis induced by DRAK2. The intracellular location of DRAK2 depended on the cell line: DRAK2 was found primarily in the nuclei of NRK, NIH3T3, and Caco-2 cells while it was present primarily in the cytoplasm of ACL-15, HeLa, and WI-38 cells. Overexpression of Myc-tagged DRAK2 led to apoptosis-like cell death in NRK cells, but not in ACL-15 cells. A GFP fusion protein of DRAK2 was spontaneously localized to the nucleus of ACL-15 cells and resulted in cell death. Nuclear localization and cell death were also observed with DRAK2(1-293) fused to the NLS of SV40 but not with DRAK2(1-293) alone. These results suggested that nuclear accumulation of DRAK2 and the resulting increase in the endogenous level of its kinase activity are required for cell death. UV irradiation caused nuclear accumulation of endogenous DRAK2 in ACL-15 cells, which was followed by apoptosis-like cell death. Knockdown of DRAK2 expression by siRNA partially suppressed UV-induced apoptosis. These results suggest that DRAK2 plays a role in UV induced apoptosis.

Streptococcus pneumoniae: The role of apoptosis in host defense and pathogenesis

Programmed cell death or apoptosis is a recognised feature of infection with Streptococcus pneumoniae, and is observed during pneumococcal meningitis and pneumonia. The cholesterol-dependent cytolysin, pneumolysin, is a major trigger of apoptosis in the brain in association with pneumococcal production of hydrogen peroxide. Pneumococcal cell wall is also an important stimulus for apoptosis. Microbial factors and host factors combine in causing apoptosis in the brain, with hippocampal neurons being particularly susceptible. In pulmonary infection epithelial cell apoptosis contributes to tissue injury but macrophage apoptosis may benefit the host, aiding microbial killing and downregulating the inflammatory response. During sepsis lymphocyte apoptosis may be harmful to the host while dendritic cell apoptosis may limit the generation of an adaptive immune response during infection. Apoptosis induction may be harmful or potentially beneficial during pneumococcal infection and understanding its function in each setting is essential to allow specific therapeutic intervention.

Role of apoptosis stimulus factor and its ligand in the induction of apoptosis in some ultraviolet induced diseases

Fas (factor of apoptosis stimulus) is one of the death receptors belonging to the tumor necrosis factor superfamily of receptors. When bound to its ligand, Fas-ligand (Fas-L), it triggers apoptosis. Ultraviolet (UV) rays can induce keratinocyte apoptosis by Fas/Fas-L interaction. The aim of the study was to evaluate the role of Fas and Fas-L in basal cell carcinoma (BCC) as an example of malignant neoplasm and discoid lupus erythematosus (DLE) as a benign skin disease, which are both induced by UV. The study included 20 cases of BCC, 20 cases of DLE and ten control cases. All biopsies of BCC and DLE were examined histopathologically. They were also examined for Fas and Fas-L by PCR. In BCC, apoptosis was detected in 60 percent of cases. Fas was found to be positive in only one case and it was found to be negative in the other 19 cases (95 %). Fas-L was found to be positive in 100 percent of cases. In DLE, apoptosis was detected in 90 percent of cases. Fas was positive in 80 percent of cases, Fas-L was positive in 90% of cases. Over-expression of Fas-L and lack of expression of Fas by tumor cells together with other factors act in favor of BCC by helping its survival and progression. Also, it seems that Fas/Fas-L interaction plays a critical role in the apoptosis seen in cases of DLE and hence in the pathogenesis of DLE.

Protein Kinase C Delta Induces Apoptosis of Vascular Smooth Muscle Cells through Induction of the Tumor Suppressor p53 by Both p38-dependent and p38-independent Mechanisms

Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. In the present study, we examined the novel PKC isoform protein kinase C delta (PKCdelta) and its role in vascular SMC apoptosis. In A10 SMCs, overexpression of PKCdelta was sufficient to induce apoptosis, whereas inhibition of PKCdelta diminished H2O2-induced apoptosis. Moreover, evidence is provided that the tumor suppressor p53 is an essential mediator of PKCdelta-induced apoptosis in SMCs. Activation of PKCdelta led to accumulation as well as phosphorylation of p53 in SMCs; this induction correlated with apoptosis. Furthermore, blocking p53 induction with small interference RNA or targeted gene deletion prevented PKCdelta-induced apoptosis, whereas restoring p53 expression rescued the ability of PKCdelta to induce apoptosis in p53 null SMCs. We also establish that PKCdelta regulates p53 at both transcriptional and post-translational levels. Specifically, the transcriptional regulation required p38 MAPK, whereas the post-translational modification, at least for serine 46, did not involve MAPK. Additionally, PKCdelta, p38 MAPK, and p53 co-associate in cells under conditions favoring apoptosis. Together, our data suggest that SMC apoptosis proceeds through a pathway that involves PKCdelta, the intermediary p38 MAPK, and the downstream target tumor suppressor p53.

83 INVITED Chemoresistance and apoptosis: shifting from the apoptotic default pathway to the adaptive stress response

Background/Purpose: Spontaneous tumor regression is a well-known characteristic in neuroblastomas. Because preliminary reports have shown that regression may be caused by apoptosis (a lethal cascade mediated by the CD95 (APO-1/Fas)-receptor), we analyzed the expression of CD95-receptors in 5 human neuroblastoma cell lines. Ceramides (known stimuli of apoptosis downstream from the CD95-receptor complex) also were used to test whether apoptosis would be induced in neuroblastoma cell cultures resistant to CD95-mediated programmed cell death. Methods: The expression of the CD95-receptor was assessed by flow cytometry after incubation with either fluorisothiocyanate-conjugated (FITC) anti-CD95-antibody (UB2) or CD95-ligand for 16 hours. Apoptotic cell death was detected via microscopy, cell viability testing (MTT, 3-[4,5 dimehylthiazole-2-yl]-2,5 diphenyltetrazoliumbromide), and flow cytometric analysis after propidium iodide staining of the DNA. Results: CD95-receptor expression was found on all neuroblastoma cell lines. Stimulation of the CD95-receptor of the malignant glioblastoma cell line LN229 (positive control) with either anti-CD95-antibody or CD95-ligand induced apoptosis. Apoptosis was not seen, however, in any of the neuroblastoma cell lines when the CD95-receptor was stimulated with anti-CD95-antibody or the CD95-ligand. Significant apoptosis was detected in all neuroblastoma cell lines after the addition of 25 μmol/L C2- and C6-ceramide. Conclusions: CD95-receptors are present on neuroblastoma cell lines, and these cells are resistant to apoptosis stimulated by anti-CD95-antibody or CD95-ligand. Apoptosis is induced, however, when these cells are treated with ceramide. A signal blockage downstream from the CD95-receptor complex and upstream of ceramide may account for this finding, and the “cellular FLICE inhibitory protein” (cFLIP) may be primarily responsible. J Pediatr Surg 35:473-479. Copyright © 2000 by W.B. Saunders Company.

A mechanism of hyperthermia‐induced apoptosis in ras‐transformed lung cells

Lung cancer, the leading cause of cancer-related deaths in both men and women, is the consequence of disordered apoptosis, induction of which may have therapeutic utility. Hyperthermia has been identified as a stimulus for apoptosis. We investigated the mechanism of hyperthermia-induced cell death in ras-transformed lung cells. Effect of hyperthermia (43 degrees C for 180 min) was compared between two cell lines, an immortalized (sv-40) normal human bronchial epithelial (BEAS2-B) and its malignant transformed (H-ras transfected) counterpart (BZR-T33). Survival after hyperthermia: 7-d growth culture BEAS2-B, 1.03 +/- 0.007 and BZR-T33, 0.39 +/- 0.008 (P < 0.05); clonogenic assays BEAS2-B, 0.76 +/- 0.003 and BZR-T33, 0.41 +/- 0.004 (P < 0.05). Hoechst positive (apoptotic) cells: BEAS2-B, 11 +/- 3% and BZR-T33, 78 +/- 5% (P < 0.05). TUNEL, DNA fragmentation, and Annexin-V all corroborate this result. Western blot comparing the effect of hyperthermia in BZR-T33 cells to BEAS2-B cells revealed: TRAIL and FAS-L displayed significant increases (threefold and twofold, respectively); caspase-3 showed a decrease in uncleaved form and an increase in cleaved form, and a 50-fold increase in activity effectively blocked with the caspase-3 inhibitor DEVD-fmk; caspase-9 showed near depletion of uncleaved; poly (ADP-ribose) polymerase (PARP) degradation was clearly visible during heating. After hyperthermia, gene expression demonstrates a 5.7-fold increase in TRAIL and insignificant changes in tumor necrosis factor-alpha (TNF-alpha), FAS-L, and caspases 3, 8, 9 in transformed cells. Data demonstrated that hyperthermia induces apoptosis in transformed cells, and that apoptosis is mediated by caspase-3 as a result of activation of cell-death membrane receptors of the tumor-necrosis-factor family. In summary, these data suggest that hyperthermia could become an additional modality in the multidisciplinary approach to the treatment of lung cancer.

Spodoptera littoralis caspase-1, a Lepidopteran effector caspase inducible by apoptotic signaling

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can successfully infect Spodoptera frugiperda SF9 cells, but in contrast, in Spodoptera littoralis SL2 cells it induces apoptosis aborting the infection. To understand better the mechanism of induction and execution of apoptosis in SL2 cells, we identified and characterized the first Spodoptera littoralis caspase, Sl-caspase-1. Sl-caspase-1 is an effector caspase that cleaves DEVD but not IETD and LEHD substrates, and the caspase-3 inhibitor DQMD-CHO inhibited this activity. It was involved in two apoptotic pathways induced by UV irradiation and virus infection. Moreover processing of Sl-caspase-1 was a determinant factor for baculovirus induction of apoptosis in SL2 cells. Since very little is known on the regulation of expression of Lepidopteran caspases, we studied Sl-caspase-1 expression after exposure to apoptosis stimuli. We found that triggering apoptosis in SL2 cells increased the steady-state level of Sl-caspase-1 without changing the level of sl-caspase-1 mRNA, suggesting that Sl-caspase-1 was post-transcriptionally up regulated. This regulation might occur as an early event in transduction of the apoptotic signal.

Myofibroblasts in Dense Model Collagen Matrices: Migration, Proliferation, Apoptosis

Fibrillar collagen matrices, that mimic the supramolecular organization of collagen in tissues, are obtained by using acid soluble collagen concentrated at 40 mg/ml and then forming fibrils at neutral pH. They present mechanical properties close to that of human dermis (1). We have previously shown that 150.000 human dermal fibroblasts seeded at the surface of collagen matrices migrate and attain a density of 5500 cells/mm3 after 28 days (2).The present work studies the phenotype of the cells migrating into dense, compared to loose (5 mg/ml) collagen networks. We demonstrate that cell migration is associated to matrix hydrolysis by MT1‐MMP on the cell front and gelatinase A (MMP2) that allow a migration distance of 350 μm after 28 days. Immunodetection of alpha‐smooth muscle actin evidences a myofibroblast phenotype for all cells penetrating both 40 and 5 mg/ml matrices. In parallel, we observe collagen I and fibronectin synthesis, typical of myofibroblasts. The TUNEL test reveals 43% of apoptotic cells in dense matrices after 28 days and a total absence of apoptosis in loose matrices. BrdU incorporation in myofibroblasts shows an intense proliferation in the dense collagen matrices that compensates cell death. Indeed the number of cells after one month of culture represents three times the number initially seeded.To conclude, in our culture conditions using 10% foetal calf serum, the stiffness of the matrix seems to be a stimulus of apoptosis; futhermore cells which express alpha‐SM actin are both able to proliferate and migrate.

Lack of alpha-synuclein does not alter apoptosis of neonatal catecholaminergic neurons.

alpha-Synuclein is an abundant neuronal protein of uncertain function linked to Parkinson's disease. Numerous studies have proposed an antiapoptotic function for alpha-synuclein, based on overexpression experiments in cell lines. To explore whether alpha-synuclein has such a physiological function, we assessed the response of wild type or alpha-synuclein null neonatal mouse sympathetic neurons to nerve growth factor deprivation, a well-characterized stimulus of neuronal apoptosis. There was no difference in the rate of neuronal loss, neuronal apoptosis, or c-jun phosphorylation. Furthermore, the absence of alpha-synuclein did not alter the magnitude of naturally occurring cell death in vivo in substantia nigra pars compacta. Therefore, alpha-synuclein is unlikely to play a significant role in apoptotic signalling in catecholaminergic neurons of the neonatal nervous system.

Apoptosis may contribute to false-positive results in the in vitro micronucleus test performed in extreme osmolality, ionic strength and pH conditions

Conditions in which clastogens produce positive responses have been increasingly challenged, and several situations have been described in which clastogenic responses would be considered not to be relevant. For example, extreme culture conditions lead to high variations of pH, osmolality or ionic strength. Apoptosis is induced in extreme culture conditions and contributes to false-positive results in the in vitro micronucleus test performed with CTLL-2 cells. These cells can enter apoptosis when exposed to apoptosis stimuli or after IL-2 deprivation, whereas the CTLL-2 Bcl2 cell line is protected from apoptosis due to the over-expression of the apoptosis inhibitor Bcl2 in bcl2- transfected CTLL-2 cells. The two cell lines were treated in extreme culture conditions of either pH or osmolality or were submitted to high ionic strength. The apoptosis level was measured in parallel with the in vitro micronucleus test using the annexin V-FITC method. Data obtained in the two cell lines suggested that apoptosis caused by extreme culture condition induces the formation of micronucleated cells, which leads to false-positive results in the in vitro micronucleus test.

Apoptosis may contribute to false-positive results in the in vitro micronucleus test performed in extreme osmolality, ionic strength and pH conditions

Conditions in which clastogens produce positive responses have been increasingly challenged, and several situations have been described in which clastogenic responses would be considered not to be relevant. For example, extreme culture conditions lead to high variations of pH, osmolality or ionic strength. Apoptosis is induced in extreme culture conditions and contributes to false-positive results in the in vitro micronucleus test performed with CTLL-2 cells. These cells can enter apoptosis when exposed to apoptosis stimuli or after IL-2 deprivation, whereas the CTLL-2 Bcl2 cell line is protected from apoptosis due to the over-expression of the apoptosis inhibitor Bcl2 in bcl2-transfected CTLL-2 cells. The two cell lines were treated in extreme culture conditions of either pH or osmolality or were submitted to high ionic strength. The apoptosis level was measured in parallel with the in vitro micronucleus test using the annexin V-FITC method. Data obtained in the two cell lines suggested that apoptosis caused by extreme culture condition induces the formation of micronucleated cells, which leads to false-positive results in the in vitro micronucleus test.

Invited commentary

The use of heat to treat disease has existed as long as recorded history. Observations relevant to cancer show heat does the following: (1) selectively kills cells as a function of thermal dose (duration of exposure × degree of temperature elevation); (2) selectively kills S-phase and radiation-resistant cells; (3) partially inhibits DNA repair; (4) kills cells with a defective heat-shock response; and (5) is a stimulus for apoptosis. Tumor cells, therefore, are more vulnerable to heat than normal cells. The clinical application of hyperthermia, often in conjunction with chemotherapeutic agents, includes three general methods based on the extent of the body treated. Localized hyperthermia utilizes heat for primary and recurrent tumors and appears most successful for head and neck tumors. Regional or isolated organ/limb hyperthermia utilizes heat restricted to specific regions of the body. Response has been documented in select patients for tumors of the limbs, liver, pancreas, esophagus, gastrointestinal tract, colon, and rectum. Whole-body hyperthermia uses methods that heat the entire body to a target core temperature of approximately 42 degrees centigrade to treat metastatic disease. Effectiveness is still controversial because of the difficulty in administering and monitoring systemic hyperthermia and limited experience.

Bax mediates the apoptosis-sensitizing effect of maspin.

Maspin, a serine protease inhibitor (serpin), can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. This may occur through maspin-mediated inhibition of pericellular proteolysis. In a recent report, we provided evidence that maspin may also suppress tumor progression by enhancing cellular sensitivity to apoptotic stimuli. To our knowledge, maspin is the only proapoptotic serpin among all of the serpins implicated thus far in apoptosis regulation. The goal of the present study is to identify the specific target molecule(s), the modification of which by maspin renders tumor cells sensitive to chemotherapeutic agents. Our cellular, molecular, and biochemical studies demonstrate an essential role of Bax in the proapoptotic effect of maspin. First, Bax was up-regulated in maspin-transfected prostate and breast tumor cells, whereas the levels of other Bcl-2 family members including Bcl-2, Bcl-xl, and Bak remained unchanged. Second, on apoptosis induction, a greater amount of Bax was translocated from cytosol to mitochondria in maspin-transfected cells. After treatment with a Bax-silencing small interfering RNA, maspin-transfected cells became significantly more resistant to drug-induced apoptosis. Consistently, the release of cytochrome c and Smac/DIABLO from mitochondria was more responsive to apoptosis stimuli in maspin-transfected cells than in the mock-transfected cells. Third, the apoptosis induction of maspin-transfected cells was associated with increased activation of both caspase-8 and caspase-9. However, a caspase-9-specific inhibitor blocked the sensitization effect of maspin in a dose-dependent and time-dependent manner, demonstrating a rate-limiting role for caspase-9. In line with the central role of the Bax-mediated mitochondrial apoptotic pathway, maspin sensitized the apoptotic response of breast and prostate carcinoma cells to various drugs, ranging from death ligands to endoplasmic reticulum stress. The link between maspin and Bax up-regulation explains the loss of maspin-expressing tumor cells in invasive breast and prostate carcinomas. Our data reveal a novel mechanism for tumor suppressive maspin and suggest that maspin may be used as a modifier for apoptosis-based cancer therapy.

Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis.

The mitochondrial uncoupling protein-3 is a member of the mitochondrial carrier protein family. As a homologue of the thermogenic brown fat uncoupling protein-1, it possesses a mitochondrial uncoupling activity and thus can influence cell energy metabolism but its exact biological function remains unclear. In the present study, uncoupling protein-3 was expressed in 293 cells using the tetracycline-inducible system and its impact on cell bioenergetics and responsiveness to the apoptotic stimulus was determined. The induction of uncoupling protein-3 expression in mitochondria did not lead to uncontrolled respiratory uncoupling in intact cells. However, it caused a GDP-inhibition of state 4 respiration and a GDP-induced re-polarization of the inner mitochondrial membrane in the presence of fatty acids, in agreement with its expected physiological behavior as an uncoupling protein (UCP). Uncoupling protein-3 expression did not cause apoptosis per se but increased the responsiveness of the cells to a mitochondrial apoptotic stimulus (i.e., addition of staurosporine in the culture medium). It enhanced caspase 3 and caspase 9 activation and favored cytochrome c release. Moreover, cells in which uncoupling protein-3 expression had been induced showed a higher mitochondrial Bax/Bcl-2 ratio essentially due to enhanced translocation of Bax from cytosol to mitochondria. Finally, the induction of uncoupling protein-3 also increased the sensitivity of mitochondria to open the permeability transition pore in response to calcium. It is concluded that the presence of uncoupling protein-3 in mitochondria sensitizes cells to apoptotic stimuli involving mitochondrial pathways.