Carmen Garrido

Hsp27 negatively regulates cell death by interacting with cytochrome c

Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative

We investigated the mechanisms of immune tolerance raised by tumors by comparing immunogenic and tolerogenic tumor cell clones isolated from a rat colon carcinoma. When injected into syngeneichosts, the immunogenic REGb cells yield tumors that are rejected, while the tolerogenic PROb cells yield progressive tumors and inhibit the regression of REGb tumors. We show here that PROb tumor volume is correlated with an expansion of CD4+CD25+ regulatory T lymphocytes in lymphoid tissues. These cells delay in vivo the rejection of REGb tumors and inhibit in vitro T cell‐mediated immune responses against REGb cells through a mechanism that requires cell contact between effector and regulatory T cells and involves TGF‐β. While total T cells fromPROb tumor‐bearing rats yield no apparent anti‐tumor immune response, depletion of CD25+ T cells restores this reactivity. A single administration of cyclophosphamide depletes CD4+CD25+ T cells in PROb tumor‐bearing animals, delays the growth of PROb tumors, and cures rats bearing established PROb tumors when followed by an immunotherapy which has no curative effect when administered alone. These results demonstrate the role of CD4+CD25+ regulatory T cells in tumor‐induced immune tolerance and the interest of regulatory T cell depletion to sensitize established tumors to immunotherapy.

Heat-shock protein 70 antagonizes apoptosis-inducing factor.

Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex. Here we show that overexpression of Hsp70 protects Apaf-1−/− cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.

Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death

Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.

Heat Shock Proteins 27 and 70: Anti-Apoptotic Proteins with Tumorigenic Properties

Heat shock proteins (HSP) HSP27 and HSP70 are expressed in response to a wide variety of physiological and environmental insults including anticancer chemotherapy, thus allowing the cell to survive to lethal conditions. Several mechanisms account for the cytoprotective effect of HSP27 and HSP70. 1) Both proteins are powerful chaperones. 2) They both inhibit key effectors of the apoptotic machinery at the pre- and post-mitochondrial level. 3) They participate in the proteasome-mediated degradation of proteins under stress conditions, thereby contributing to the so called “protein triage”. In cancer cells, the expression of HSP27 and/or HSP70 is abnormally high, and both HSP27 and HSP70 may participate in oncogenesis and in resistance to chemotherapy. In rodent models, HSP27 or HSP70 over-expression increases tumor growth and metastatic potential. The depletion or inhibition of HSP27 and HS70 frequently reduces the size of the tumors and even can cause their complete involution (for HSP70). Therefore, the inhibition of HSP70 and HSP27 has become a novel strategy of cancer therapy.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.

HSP27 inhibits cytochrome c-dependent activation of procaspase-9

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.—Garrido, C., Bruey, J.‐M., Fromentin, A., Hammann, A., Arrigo, A. P., Solary, E. HSP27 inhibits cytochrome c‐dependent activation of procaspase‐9. FASEB J. 13, 2061–2070 (1999)

Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy

Stress or heat shock proteins (HSPs) are the most conserved proteins present in both prokaryotes and eukaryotes. Their expression is induced in response to a wide variety of physiological and environmental insults. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and sress‐accumulated misfolded proteins, and preventing their aggregation. HSPs have a dual function depending on their intracellular or extracellular location. Intracellular HSPs have a protective function. They allow the cells to survive lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several HSPs have also been demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream and downstream of the mitochondrial events. On the other hand, extracellular located or membrane‐bound HSPs mediate immunological functions. They can elicit an immune response modulated either by the adaptive or innate immune system. This review will focus on HSP27, HSP70, and HSP90. We will discuss the dual role of these HSPs, protective vs. immunogenic properties, making a special emphasis in their utility as targets in cancer therapy.

Heat shock proteins: essential proteins for apoptosis regulation.

•  Introduction •  Main HSPs •  HSPs, cell signalling and apoptosis ‐  HSPs’targets in upstream signalling pathways ‐  HSPs’targets at the mitochondrial level ‐  HSPs’targets at the post‐mitochondrial level ‐  HSPs and the extrinsic death receptor pathway •  HSPs and alternatives, caspase‐independent, apoptosis‐like pathways •  What directs the interaction of a HSP with a given apoptotic partner? •  Role of HSPs in haematological malignancies •  HSPs inhibitors: drugs of the future?