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Cohen syndrome (CS) is a rare genetic disorder due to mutations in VPS13B gene. Among various clinical and biological features, CS patients suffer from inconsistent neutropenia, which is associated with recurrent but minor infections. We demonstrate here that this neutropenia results from an exaggerate rate of neutrophil apoptosis. Besides this increased cell death, which occurs in the absence of any endoplasmic reticulum stress or defect in neutrophil elastase (ELANE) expression or localization, all neutrophil functions appeared to be normal. We showed a disorganization of the Golgi apparatus in CS neutrophils precursors, that correlates with an altered glycosylation of ICAM-1 in these cells, as evidenced by a migration shift of the protein. Furthermore, a striking decrease in the expression of SERPINB1 gene, which encodes a critical component of neutrophil survival, was detected in CS neutrophils. These abnormalities may account for the excessive apoptosis of neutrophils leading to neutropenia in CS. KEY MESSAGES: Cohen syndrome patients' neutrophils display normal morphology and functions. Cohen syndrome patients' neutrophils have an increased rate of spontaneous apoptosis compared to healthy donors' neutrophils. No ER stress or defective ELA2 expression or glycosylation was observed in Cohen syndrome patients' neutrophils. SerpinB1 expression is significantly decreased in Cohen syndrome neutrophils as well as in VPS13B-deficient cells.

IF 5.008; International audience; Gap junctional communication between cancer cells and blood capillary cells is crucial to tumor growth and invasion. Gap junctions may transfer microRNAs (miRs) among cells. Here, we explore the impact of such a transfer in co-culture assays, using the antitumor miR-145 as an example. The SW480 colon carcinoma cells form functional gap junction composed of connexin-43 (Cx43) with human microvascular endothelial cells (HMEC). When HMEC are loaded with miR-145-5p mimics, the miR-145 level drastically increases in SW480. The functional inhibition of gap junctions, using either a gap channel blocker or siRNA targeting Cx43, prevents this increase. The transfer of miR-145 also occurs from SW480 to HMEC but not in non-contact co-cultures, excluding the involvement of soluble exosomes. The miR-145 transfer to SW480 up-regulates their Cx43 expression and inhibits their ability to promote angiogenesis. Our results indicate that the gap junctional communication can inhibit tumor growth by transferring miRs from one endothelial cell to neighboring tumor cells. This "bystander" effect could find application in cancer therapy.

IF 7.932; International audience; Heat shock protein 110 (HSP110) is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps cells survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently shown that colorectal cancer patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110-inactivating mutation (HSP110DE9). In this work, we used patient biopsies, human colorectal cancer cells grown in vitro and in vivo (xenografts), and intestinal crypts to demonstrate that HSP110 is also involved in colon cancer growth. We showed that HSP110 induces colon cancer cell proliferation and that this effect is associated with STAT3 activation, specifically an increase in STAT3 phosphorylation, nuclear translocation and transcription factor activity. STAT3 inhibition blocks the proliferative effect of HSP110. From a molecular standpoint, we demonstrated that HSP110 directly binds to STAT3, thereby facilitating its phosphorylation by JAK2. Finally, we showed a correlation between HSP110 expression and STAT3 phosphorylation in colon cancer patient samples. Thus, the expression of HSP110 in colon cancer contributes to STAT3-dependent tumor growth and the frequent inactivating mutation of this chaperone is probably an important event underlying the improved prognosis in colon cancer displaying MSI.

IF 5.008; International audience; Extensive invasion and angiogenesis are hallmark features of malignant glioblastomas. Here, we co-cultured U87 human glioblastoma cells and human microvascular endothelial cells (HMEC) to demonstrate the exchange of microRNAs that initially involve the formation of gap junction communications between the two cell types. The functional inhibition of gap junctions by carbenoxolone blocks the transfer of the anti-tumor miR-145-5p from HMEC to U87, and the transfer of the pro-invasive miR-5096 from U87 to HMEC. These two microRNAs exert opposite effects on angiogenesis in vitro. MiR-5096 was observed to promote HMEC tubulogenesis, initially by increasing Cx43 expression and the formation of heterocellular gap junctions, and secondarily through a gap-junction independent pathway. Our results highlight the importance of microRNA exchanges between tumor and endothelial cells that in part involves the formation of functional gap junctions between the two cell types.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by myofibroblast proliferation. Transition of epithelial/mesothelial cells into myofibroblasts [epithelial-to-mesenchymal transition (EMT)] occurs under the influence of transforming growth factor (TGF)-β1, with Snail being a major transcription factor. We study here the role of the heat-shock protein HSP27 in fibrogenesis and EMT. In vitro, we have up- and down-modulated HSP27 expression in mesothelial and epithelial cell lines and studied the expression of different EMT markers induced by TGF-β1. In vivo, we inhibited HSP27 with the antisense oligonucleotide OGX-427 (in phase II clinical trials as anticancer agent) in our rat subpleural/pulmonary fibrosis models. We demonstrate that HSP27 is strongly expressed during the fibrotic process in patients with IPF and in different in vivo models. We showed that HSP27 binds to and stabilizes Snail and consequently induces EMT. Conversely, HSP27 knockdown leads to Snail proteasomal degradation, thus inhibiting TGF-β1-induced EMT. Inhibition of HSP27 with OGX-427 efficiently blocks EMT and fibrosis development. Controls in vivo were an empty adenovirus that did not induce fibrosis and a control antisense oligonucleotide. The present work opens the possibility of a new therapeutic use for HSP27 inhibitors against IPF, for which there is no conclusively effective treatment.

International audience; Background: Exosomes, via heat shock protein 70 (HSP70) expressed in their membrane, are able to interact with the toll-like receptor 2 (TLR2) on myeloid-derived suppressive cells (MDSCs), thereby activating them.Methods: We analyzed exosomes from mouse (C57Bl/6) and breast, lung, and ovarian cancer patient samples and cultured cancer cells with different approaches, including nanoparticle tracking analysis, biolayer interferometry, FACS, and electron microscopy. Data were analyzed with the Student's t and Mann-Whitney tests. All statistical tests were two-sided.Results: We showed that the A8 peptide aptamer binds to the extracellular domain of membrane HSP70 and used the aptamer to capture HSP70 exosomes from cancer patient samples. The number of HSP70 exosomes was higher in cancer patients than in healthy donors (mean, ng/mL +/- SD = 3.5 +/- 1.7 vs 0.17 +/- 0.11, respectively, P = .004). Accordingly, all cancer cell lines examined abundantly released HSP70 exosomes, whereas "normal" cells did not. HSP70 had higher affinity for A8 than for TLR2; thus, A8 blocked HSP70/TLR2 association and the ability of tumor-derived exosomes to activate MDSCs. Treatment of tumor-bearing C57Bl/6 mice with A8 induced a decrease in the number of MDSCs in the spleen and inhibited tumor progression (n = 6 mice per group). Chemotherapeutic agents such as cisplatin or 5FU increase the amount of HSP70 exosomes, favoring the activation of MDSCs and hampering the development of an antitumor immune response. In contrast, this MDSC activation was not observed if cisplatin or 5FU was combined with A8. As a result, the antitumor effect of the drugs was strongly potentiated.Conclusions: A8 might be useful for quantifying tumor-derived exosomes and for cancer therapy through MDSC inhibition.

When overexpressed, the stress protein heat shock protein 70 (HSP70) increases the oncogenic potential of cancer cells in rodent models. HSP70 also prevents apoptosis, thereby increasing the survival of cells exposed to a wide range of otherwise lethal stimuli. These protective functions of HSP70 involve its interaction with and neutralization of the adaptor molecule apoptotic protease activation factor-1, implicated in caspase activation, and the flavoprotein apoptosis-inducing factor (AIF), involved in caspase-independent cell death. We have shown previously that a peptide containing the AIF sequence involved in its interaction with HSP70 (ADD70, amino acids 150-228) binds to and neutralizes HSP70 in the cytosol, thereby sensitizing cancer cells to apoptosis induced by a variety of death stimuli. Here, we show that expression of ADD70 in tumor cells decreases their tumorigenicity in syngeneic animals without affecting their growth in immunodeficient animals. ADD70 antitumorigenic effects are associated with an increase in tumor-infiltrating cytotoxic CD8+ T cells. In addition, ADD70 sensitizes rat colon cancer cells (PROb) and mouse melanoma cells (B16F10) to the chemotherapeutic agent cisplatin. ADD70 also shows an additive effect with HSP90 inhibition by 17-allylamino-17-demethoxygeldanamycin in vitro. Altogether, these data indicate the potential interest of targeting the HSP70 interaction with AIF for cancer therapy. (Cancer Res 2006; 66(8): 4191-7)

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.

International audience; The proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs.

Procaspase-2 is one of the cysteine aspartate proteases involved in apoptotic cell death. Alternative splicing of CASP-2 messenger RNA generates a long isoform, procaspase-2L, whose overexpression induces cell death and a truncated isoform, procaspase-2S, whose function remains poorly defined. The present study explored the consequences of procaspase-2S overexpression in U937 human leukemic cells exposed to the topoisomerase II inhibitor etoposide as an apoptotic stimulus. Overexpression of procaspase-2S in U937 cells partially prevented nuclear changes associated with etoposide-induced cell death, as determined by Hoechst 33342 staining of nuclear chromatin and electron microscopy studies. Procaspase-2S also prevented the maturation of apoptotic bodies, delayed phosphatidylserine externalization on the plasma membrane and prevented the cleavage and activation of procaspase-2L. These effects were not observed when the cysteine 289 in the consensus QACRG motif was mutated into a serine. Wild-type procaspase-2S overexpression did not influence the cleavage of procaspase-3, procaspase-7 and poly(ADP-ribose)polymerase nor the fragmentation of nuclear DNA into nucleosome-sized fragments. Altogether, these results indicate that the short isoform of procaspase-2 negatively interferes with selective features of apoptosis, an activity that is suppressed by mutation of the cysteine 289.

Stress-inducible HSP27 protects cells from death through various mechanisms. We have recently demonstrated that HSP27 can also enhance the degradation of some proteins through the proteasomal pathway. Here, we show that one of these proteins is the cyclin-dependent kinase (Cdk) inhibitor p27Kip1. The ubiquitination and degradation of this protein that favors progression through the cell cycle was previously shown to involve either a Skp2-dependent mechanism,i.e., at the S-/G2-transition, or a KPC (Kip1 ubiquitination-promoting complex)-dependent mechanism, i.e.,at the G0/G1 transition. In this work, we demonstrate that, in response to serum depletion, p27Kip1 cellular content first increases then progressively decreases as cells begin to die. In this stressful condition, HSP27favors p27Kip1 ubiquitination and degradation by the proteasome. A similar observation was made in response to stress induced by the NO donor glyceryl trinitrate (GTN). HSP27-mediated ubiquitination ofp27Kip1 does not require its phosphorylation on Thr187 or Ser-10, nor does it depend on the SCFSkp2 ubiquitin ligase E3 complex. It facilitates the G1/S transition,which suggests that, in stressful conditions, HSP27might render quiescent cells competent to re-enter the cell cycle.

To investigate the role of the small 27-kDa heat-shock protein (Hsp27) in the intrinsic resistance of colon cancer cells to doxorubicin, we modified Hsp27 expression either genetically by transfection or pharmacologically by cisplatin treatment. HT-29 cells were transfected with a full-length Hsp27 construct in the sense or antisense orientation. We found a good correlation between cell survival after doxorubicin treatment and Hsp27 content. A similar correlation was found for the thermoresistance of the Hsp27-transfected cells. In contrast, the sensitivity of the different transfected cells to 5-fluorouracil was not modified. cis-Platinum(II)diammine dichloride (cisplatin) treatment of HT-29 or Caco2 cells dramatically increased their Hsp27 mRNA and protein content. Accordingly, the cells became thermoresistant. Contrary to what has been previously assumed, however, cell resistance to doxorubicin was reduced. Our data suggest that the decreased resistance of the cells to doxorubicin may be due to a concomitant increase of topoisomerase II expression, the main target of anthracyclines. In conclusion, although Hsp27 seems to participate in the natural resistance of colon cancer cells to anthracyclines, its increase after cisplatin treatment is not associated with a decreased cytotoxicity to doxorubicin.

Inactivation of p53 found in more than half of human cancers is often associated with increased tumor resistance to anti-cancer therapy. We have previously shown that overexpression of the phosphatase Wip1 in p53-negative tumors sensitizes them to chemotherapeutic agents, while protecting normal tissues from the side effects of anti-cancer treatment. In this study, we decided to search for kinases that prevent Wip1-mediated sensitization of cancer cells, thereby interfering with efficacy of genotoxic anti-cancer drugs. To this end, we performed a flow cytometry-based screening in order to identify kinases that regulated the levels of γH2AX, which were used as readout. Another criterion of the screen was increased sensitivity of p53-negative tumor cells to cisplatin (CDDP) in a Wip1-dependent manner. We have found that a treatment with a low dose (75 nM) of MK-1775, a recently described specific chemical inhibitor of Wee1, decreases CDDP-induced H2AX phosphorylation in p53-negative cells and enhances the Wip1-sensitization of p53-negative tumors. We were able to reduce CDDP effective concentration by 40% with a combination of Wip1 overexpression and Wee1 kinase inhibition. We have observed that Wee1 inhibition potentiates Wip1-dependent tumor sensitization effect by reducing levels of Hipk2 kinase, a negative regulator of Wip1 pathway. In addition, during CDDP treatment, the combination of Wee1 inhibition and Wip1 overexpression has a mild but significant protective effect in normal cells and tissues. Our results indicate that inhibition of the negative regulators of Wip1 pathway, Wee1 and Hipk2, in p53-negative tumors could potentiate efficiency of chemotherapeutic agents without concomitant increase of cytotoxicity in normal tissues. The development and clinical use of Wee1 and Hipk1 kinase chemical inhibitors might be a promising strategy to improve anti-cancer therapy.

The inactivation of the p53 tumor suppressor pathway in many cancers often increases their resistance to anticancer therapy. Here we show that a previously proposed strategy directed to Wip1 inhibition could be ineffective in tumors lacking p53. On the contrary, Wip1 overexpression sensitized these tumors to chemotherapeutic agents. This effect was mediated through interaction between Wip1 and RUNX2 that resulted, in response to anticancer treatment, in RUNX2-dependent transcriptional induction of the proapoptotic Bax protein. The potentiating effects of Wip1 overexpression on chemotherapeutic agents were directed only to tumor cells lacking p53. The overexpression of Wip1 in normal tissues provided protection from cisplatin-induced apoptosis through decreased strength of upstream signaling to p53. Thus, Wip1 phosphatase promotes apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.

International audience; The inhibitor of apoptosis protein cIAP1 (cellular inhibitor of apoptosis protein-1) is a potent regulator of the tumor necrosis factor (TNF) receptor family and NF-B signaling pathways in the cytoplasm. However, in some primary cells and tumor cell lines, cIAP1 is expressed in the nucleus, and its nuclear function remains poorly understood. Here, we show that the N-terminal part of cIAP1 directly interacts with the DNA binding domain of the E2F1 transcription factor. cIAP1 dramatically increases the transcriptional activity of E2F1 on synthetic and CCNE promoters. This function is not conserved for cIAP2 and XIAP, which are cytoplasmic proteins. Chromatin immunoprecipitation experiments demonstrate that cIAP1 is recruited on E2F binding sites of the CCNE and CCNA promoters in a cell cycle-and differentiation-dependent manner. cIAP1 silencing inhibits E2F1 DNA binding and E2F1-mediated transcriptional activation of the CCNE gene. In cells that express a nuclear cIAP1 such as HeLa, THP1 cells and primary human mammary epithelial cells, down-regulation of cIAP1 inhibits cyclin E and A expression and cell proliferation. We conclude that one of the functions of cIAP1 when localized in the nucleus is to regulate E2F1 transcriptional activity.

The inhibition of heat shock protein 70 (HSP70) is an emerging strategy in cancer therapy. Unfortunately, no specific inhibitors are clinically available. By yeast two-hybrid screening, we have identified multiple peptide aptamers that bind HSP70. When expressed in human tumor cells, two among these peptide aptamers—A8 and A17—which bind to the peptide-binding and the ATP-binding domains of HSP70, respectively, specifically inhibited the chaperone activity, thereby increasing the cells' sensitivity to apoptosis induced by anticancer drugs. The 13-amino acid peptide from the variable region of A17 (called P17) retained the ability to specifically inhibit HSP70 and induced the regression of subcutaneous tumors in vivo after local or systemic injection. This antitumor effect was associated with an important recruitment of macrophages and T lymphocytes into the tumor bed. Altogether, these data indicate that peptide aptamers or peptides that target HSP70 may be considered as novel lead compounds for cancer therapy. Cancer Res; 71(2); 484–95. ©2011 AACR.

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.

Transcription intermediary factor 1γ (TIF1γ) was suggested to play a role in erythropoiesis. However, how TIF1γ regulates the development of different blood cell lineages and whether TIF1γ is involved in human hematological malignancies remain to be determined. Here we have shown that TIF1γ was a tumor suppressor in mouse and human chronic myelomonocytic leukemia (CMML). Loss of Tif1g in mouse HSCs favored the expansion of the granulo-monocytic progenitor compartment. Furthermore, Tif1g deletion induced the age-dependent appearance of a cell-autonomous myeloproliferative disorder in mice that recapitulated essential characteristics of human CMML. TIF1γ was almost undetectable in leukemic cells of 35% of CMML patients. This downregulation was related to the hypermethylation of CpG sequences and specific histone modifications in the gene promoter. A demethylating agent restored the normal epigenetic status of the TIF1G promoter in human cells, which correlated with a reestablishment of TIF1γ expression. Together, these results demonstrate that TIF1G is an epigenetically regulated tumor suppressor gene in hematopoietic cells and suggest that changes in TIF1γ expression may be a biomarker of response to demethylating agents in CMML.

Heat shock protein 27 (HSP27) is a chaperone whose cellular expression increases in response to various stresses and protects the cell either by inhibiting apoptotic cell death or by promoting the ubiquitination and proteasomal degradation of specific proteins. Here, we show that globin transcription factor 1 (GATA-1) is a client protein of HSP27. In 2 models of erythroid differentiation; that is, in the human erythroleukemia cell line, K562 induced to differentiate into erythroid cells on hemin exposure and CD34+ human cells ex vivo driven to erythroid differentiation in liquid culture, depletion of HSP27 provokes an accumulation of GATA-1 and impairs terminal maturation. More specifically, we demonstrate that, in the late stages of the erythroid differentiation program, HSP27 is phosphorylated in a p38-dependent manner, enters the nucleus, binds to GATA-1, and induces its ubiquitination and proteasomal degradation, provided that the transcription factor is acetylated. We conclude that HSP27 plays a role in the fine-tuning of terminal erythroid differentiation through regulation of GATA-1 content and activity.

A monoclonal antibody, F11C, was raised against rat colon cancer cells. Its immunoreactivity on normal human and rat gut as well as human and rat colonic tumours was studied by the avidin-biotin-peroxidase complex technique. In both normal rat and human gastrointestinal tract, F11C stained surface epithelial cells from the fundus to distal colon, mainly as supranuclear vesicles. These vesicles appeared to be part of the Golgi apparatus on electron microscopy with immunogold labelling. Twenty primary rat colon tumours and 28 of 43 human colon tumours were also stained, with a heterogeneous pattern but much more strongly than the normal colonic mucosa. Biochemical purification suggested that in rat tumours F11C epitope was carried by a high molecular weight glycoprotein. Absorption experiments with synthetic oligosaccharides showed that F11C monoclonal antibody reacted with blood group A-related oligosaccharides. Nevertheless, F11C reactivity on human tissues was not related to the individual ABO or Lewis phenotype.

Heat shock protein 27 (HSP27) accumulates in stressed cells and helps them to survive adverse conditions. We have already shown that HSP27 has a function in the ubiquitination process that is modulated by its oligomerization/phosphorylation status. Here, we show that HSP27 is also involved in protein sumoylation, a ubiquitination-related process. HSP27 increases the number of cell proteins modified by small ubiquitin-like modifier (SUMO)-2/3 but this effect shows some selectivity as it neither affects all proteins nor concerns SUMO-1. Moreover, no such alteration in SUMO-2/3 conjugation is achievable by another HSP, such as HSP70. Heat shock factor 1 (HSF1), a transcription factor responsible for HSP expression, is one of the targets of HSP27. In stressed cells, HSP27 enters the nucleus and, in the form of large oligomers, binds to HSF1 and induces its modification by SUMO-2/3 on lysine 298. HSP27-induced HSF1 modification by SUMO-2/3 takes place downstream of the transcription factor phosphorylation on S303 and S307 and does not affect its DNA-binding ability. In contrast, this modification blocks HSF1 transactivation capacity. These data show that HSP27 exerts a feedback inhibition of HSF1 transactivation and enlighten the strictly regulated interplay between HSPs and HSF1. As we also show that HSP27 binds to the SUMO-E2-conjugating enzyme, Ubc9, our study raises the possibility that HSP27 may act as a SUMO-E3 ligase specific for SUMO-2/3.

To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM).U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei.FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained.QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.

Heat shock protein-27 (HSP27) and alphaB-crystallin are ubiquitous small heat shock proteins whose expression is induced in response to a wide variety of physiological and environmental insults. They allow the cells to survive in otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of these small heat shock proteins. First, these proteins are powerful molecular chaperones whose main function is to prevent the aggregation of nascent and stress-accumulated misfolded proteins. Second, they interact directly with various components of the tightly regulated programmed cell death machinery, upstream and downstream of the mitochondrial events. Third, they appear to play a role in the proteasome-mediated degradation of selected proteins. Both HSP27 and alphaB-crystallin were also proposed to participate in the development of neurodegenerative diseases and malignant tumors in which their overexpression could induce drug resistance. Altogether, these properties suggest that these small heat shock proteins are appropriate targets for modulating cell death pathways.

We have shown previously that the death receptor CD95 could contribute to anticancer drug-induced apoptosis of colon cancer cells. In addition, anticancer drugs cooperate with CD95 cognate ligand or agonistic antibodies to trigger cancer cell apoptosis. In the present study, we show that the anticancer drug cisplatin induces clustering of CD95 at the surface of the human colon cancer cell line HT29, an event inhibited by the inhibitor of acid sphingomyelinase (aSMase) imipramine. The cholesterol sequestering agent nystatin also prevents cisplatin-induced CD95 clustering and decreases HT29 cell sensitivity to cisplatin-induced apoptosis and the synergy between cisplatin and anti-CD95 agonistic antibodies. CD95, together with the adaptor molecule Fas-associated death domain and procaspase-8, is redistributed into cholesterol- and sphingolipid-enriched cell fractions after cisplatin treatment, suggesting plasma membrane raft involvement. Interestingly, nystatin prevents the translocation of the aSMase to the extracellular surface of plasma membrane and the production of ceramide, suggesting that these early events require raft integrity. In addition, nystatin prevents cisplatin-induced transient increase in plasma membrane fluidity that could be required for CD95 translocation. Together, these results demonstrate that cisplatin activates aSMase and induces ceramide production, which triggers the redistribution of CD95 into the plasma membrane rafts. Such redistribution contributes to cell death and sensitizes tumor cells to CD95-mediated apoptosis.

We and others have previously reported in an in vivo rat colon cancer cell model that cell death precedes and is necessary for the development of a specific antitumoral immune response. To sensitize colon cancer cells to death, we depleted cytochrome c by stable transfection with an antisense construct. Cytochrome c depletion sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli. This increased sensitization to a necrosis-like cell death may be related to a decrease in cellular ATP levels and an increase in reactive oxygen species production caused by cytochrome c depletion. In vivo, depletion of cytochrome c decreases the tumorigenicity of colon cancer cells in syngeneic rats without influencing their growth in immune-deficient animals. Furthermore, decreased expression of cytochrome c in tumor cells facilitates in vivo “necrotic” cell death and the induction of a specific immune response. These results delineate a novel strategy to sensitize colon cancer cells to chemotherapy and to increase their immunogenicity in immuno-competent hosts.

Background & Aims: We have previously reported the role of nitric oxide in colon tumor regression in vivo. The present study was designed to explore the influence of an endogenous nitric oxide donor, glyceryl trinitrate (GTN), on cell death pathways in colon cancer cells. Methods: Human colon cancer cell lines were treated with the NO donor GTN. Apoptosis was identified by morphological criteria and the terminal deoxynucleotidyl transferase–mediated deoxyuridine (TUNEL) method. The mitochondrial transmembrane potential was studied by flow cytometry, cytochrome c release by Western blot, and caspase activation by combining fluorogenic peptide substrates, peptide inhibitors, and immunoblotting. Expression of death receptors was studied by flow cytometry and confocal microscopy. Results: GTN induces a dose- and time-dependent cell death by apoptosis in colon cancer cells. This cell death pathway involves the mitochondria and caspases, mainly caspase-1 and caspase-10. In contrast, caspase-3 activation is a late and limited event. Death receptors are not involved in GTN-mediated cell death, while GTN sensitizes tumor cells to Fas-ligand–induced apoptosis. This permissive effect correlates with an increased expression of Fas receptor and a decreased expression of several endogenous inhibitors of apoptosis (IAPs). Conclusions: Our results indicate that GTN (1) activates an unusual caspase cascade to induce apoptosis in colon cancer cells and (2) sensitizes these cells to Fas-mediated cell death by increasing the expression of Fas and decreasing the expression of several IAPs. GASTROENTEROLOGY 2002;123:235-246

Engagement of the plasma membrane receptor Fas can induce apoptosis of leukemic cells. Signaling through Fas requires the formation of a death-inducing signaling complex (DISC) that involves the cytoplasmic domain of Fas, the adaptor molecule FADD/MORT-1, and procaspase-8. The present study investigated whether another caspase, known as procaspase-2L, played a role in Fas-mediated cell death. A series of human leukemic variant cells was derived by stable transfection with aCASP2L antisense construct (CASP2L/AS).Specific down-regulation of procaspase-2L decreased the sensitivity of these cells to apoptosis induced by an agonistic anti-Fas antibody (Ab, clone CH11), as determined by studying DNA fragmentation, chromatin condensation, and externalization of phosphatidylserine on the plasma membrane. In leukemic cells transfected with an empty vector, anti-Fas Ab treatment activated caspase-8, decreased the expression of the BH3 domain-only protein Bid, triggered the release of cytochrome c from the mitochondria to the cytosol, and activated caspase-3. All these events could not be observed when CASP2L/AS cells were similarly treated with anti-Fas Abs. CASP2L/AStransfection did not inhibit the formation of the DISC and no direct interaction between procaspase-2L and either Fas or FADD or procaspase-8 was identified. Down-regulation of procaspase-2L inhibited anti-Fas Ab–mediated cleavage of c-FLIP (FLICE-inhibitory protein), a protein that interferes with the formation of a functional DISC. These results suggest that the long isoform of caspase-2 plays a role in the Fas-mediated pathway to cell death by contributing to caspase-8 activation at the DISC level.

The counterattack hypothesis, suggesting that cancer cells express Fas ligand (FasL) and are able to kill Fas-expressing tumor-infiltrating activated T cells, was supported by reports of the killing of Jurkat cells by FasL-expressing human colon cancer cell lines. Through the use of an improved cytotoxic assay in which soluble FasL and FasL-transfected KFL9 cells were used as positive controls, we show that none of seven human colon cancer cell lines induce apoptosis of two Fas-expressing target cell lines, Jurkat and L1210-Fas cells. Moreover, in coculture experiments, cancer cell monolayers do not inhibit the growth of Fas-expressing lymphoid cells. Although FasL mRNA and protein were detected in the extracts of the colon cancer cell lines, flow cytometry and confocal microscopy failed to detect the protein on the surface of tumor cells. These results suggest that the counterattack of tumor-infiltrating T lymphocytes by cancer cells may not account for immune tolerance toward tumor cells.

We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.

We have previously shown that treatment by anticancer drugs sensitized tumor cells to Fas (APO-1/CD95)-mediated cell death. The present study demonstrates that the cytotoxic drugs cisplatin, doxorubicin and mitomycin C induce the accumulation of the Fas receptor, the FADD adaptor molecule, the procaspases-8, -3 and -2L and the proapoptotic molecule Bax in several human colon cancer cells. This upregulation is also observed in U3A myeloblastoma cells that do not express STAT-1, a transcription factor involved in the constitutive expression of procaspases. We conclude that anticancer drugs sensitize tumor cells to Fas-mediated cell death by a STAT-1-independent upregulation of molecules involved in this apoptotic pathway.

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.

We have previously shown that growth of HT29 human colorectal cancer cells at confluence increased their resistance to the cytotoxic agent cisplatin. This study further explores the mechanisms of this resistance phenotype. DNA platination induced by cisplatin exposure is slightly reduced by confluence. However, at an equivalent DNA platination level, non-confluent cells accumulate in the G2/M phase of the cell cycle, demonstrate aberrant mitotic figures and die by apoptosis, while confluent cells progress slowly through the cell cycle, do not reach mitosis and are more resistant to drug-induced cell death. At a molecular level, cisplatin enhances cyclin B and p34cdc2 levels and histone H1 kinase activity in non-confluent, but not in confluent, cells. Furthermore, when HT29 cells reach confluence, expression of the cyclin-dependent kinase inhibitor p27Kip1 increases and cells accumulate in the G0/G1 phase of the cell cycle. Transfection-mediated over-expression of p27Kip1 in non-confluent HT29 cells decreases the cytotoxic activity of cisplatin as well as its ability to trigger apoptosis. Non-confluent HT29 cells over-expressing p27Kip1 are also more resistant to doxorubicin, etoposide and 5-fluorouracil. Our results suggest that p27Kip1 contributes to the confluence-dependent resistance phenotype. Int. J. Cancer 77:796–802, 1998. © 1998 Wiley-Liss, Inc.

Background The transmembrane receptor Fas, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death (i.e., apoptosis). Fas and Fas ligand also influence the ability of cytotoxic T lymphocytes and natural killer cells to eliminate tumor cells. However, by inducing apoptosis in activated T cells, the Fas/Fas ligand system may protect some tumor cells from clearance by the immune system. Anticancer drugs enhance Fas ligand expression on the surface of Fas receptor-expressing leukemia cells, thus suggesting that apoptosis caused by these drugs may be mediated via the Fas/Fas ligand system. Purpose This study was conducted to further investigate the relationship between the modulation of Fas receptor gene and protein expression by treatment of cells with cytotoxic drugs and the immune clearance of tumor cells. Methods Fas expression on human HT29 colon carcinoma cells treated with a variety of anticancer drugs (cisplatin, doxorubicin, mitomycin C, fluorouracil, and camptothecin) was analyzed by use of quantitative flow cytometry. Human HCT8R and HCT116 colon carcinoma cells and human U937 leukemia cells were treated with cisplatin only and analyzed in the same way. Fas ligand messenger RNA and protein levels were studied by use of a reverse transcription-polymerase chain reaction assay and by flow cytometry. Fas gene expression and messenger RNA levels in cisplatin-treated HT29 cells were characterized by use of in vitro nuclear run-on and northern blot hybridization assays. The cytotoxic activities of agonistic anti-Fas antibodies, Fas ligand, and allogeneic peripheral blood leukocytes, in the absence or presence of Fas-blocking monoclonal antibodies, against tumor cells were assessed by methylene blue staining and chromium-51 release assays. Results Clinically relevant concentrations of cisplatin, doxorubicin, mitomycin C, fluorouracil, or camptothecin enhanced Fas receptor expression on the plasma membrane of HT29 cells. Cisplatin-mediated increases in Fas expression were confirmed in HCT8R, HCT116, and U937 cells. The enhancement of Fas protein expression was associated with an increased sensitivity of cisplatin-treated tumor cells to agonistic anti-Fas antibodies, to soluble Fas ligand, and to allogeneic peripheral blood leukocyte-mediated cytotoxicity. Each of these effects was blocked by co-treatment of the cells with antagonistic anti-Fas antibody. Conclusion and implications In addition to their direct cytotoxic effects, chemotherapeutic drugs sensitize tumor cells to Fas-mediated cytotoxicity and Fas-dependent immune clearance. On the basis of these findings, new strategies might be developed to improve the efficacy of these drugs.