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The Carboxy-Terminal Modulator Protein (CTMP) protein was identified as a PKB inhibitor that binds to its hydrophobic motif. Here, we report mitochondrial localization of endogenous and exogenous CTMP. CTMP exhibits a dual sub-mitochondrial localization as a membrane-bound pool and a free pool of mature CTMP in the inter-membrane space. CTMP is released from the mitochondria into the cytosol early upon apoptosis. CTMP overexpression is associated with an increase in mitochondrial membrane depolarization and caspase-3 and polyADP-ribose polymerase (PARP) cleavage. In contrast, CTMP knock-down results in a marked reduction in the loss of mitochondrial membrane potential as well as a decrease in caspase-3 and PARP activation. Mutant CTMP retained in the mitochondria loses its capacity to sensitize cells to apoptosis. Thus, proper maturation of CTMP is essential for its pro-apoptotic function. Finally, we demonstrate that CTMP delays PKB phosphorylation following cell death induction, suggesting that CTMP regulates apoptosis via inhibition of PKB.

The mechanisms through which regulatory T cells accumulate in lymphoid organs of tumor-bearing hosts remain elusive. Our experiments indicate that the accumulation of CD4+CD25+ regulatory T cells (T reg cells) expressing FoxP3 and exhibiting immunosuppressive function originates from the proliferation of naturally occurring CD25+ T cells and requires signaling through transforming growth factor (TGF)–β receptor II. During tumor progression, a subset of dendritic cells (DCs) exhibiting a myeloid immature phenotype is recruited to draining lymph nodes. This DC subset selectively promotes the proliferation of T reg cells in a TGF-β–dependent manner in mice and rats. Tumor cells are necessary and sufficient to convert DCs into regulatory cells that secrete bioactive TGF-β and stimulate T reg cell proliferation. In conclusion, tumor expansion can stimulate T reg cells via a specific DC subset.

Stress or heat shock proteins (HSPs) are ubiquitous and highly conserved proteins whose expression is induced in response to a wide variety of physiological and environmental insults. They allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. These proteins play an essential role in intracellular "house-keeping" by assisting the correct folding of nascent and stress-accumulated misfolded proteins and preventing their aggregation. Several HSPs have also demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream, and downstream of the mitochondrial events. Finally, HSPs could play a role in the proteasome-mediated degradation of selected proteins under stress conditions. Altogether, these properties could make HSPs appropriate targets for modulating cell death pathways.

REGb cell line, a highly immunogenic tumor cell variant isolated from a rat colon cancer, yields regressive tumors when injected into syngeneic hosts. We previously demonstrated that REGb tumor immunogenicity was related to the capacity of releasing dead cells in vivo. Also, in vitro, REGb cell monolayers release dead cells, especially when cultured in serum-free medium. In the current study, we show that the release of dead cells results from an atypical death process associating features of necrosis and apoptosis. In spite of features considered as hallmarks of caspase-dependent apoptosis, including chromatin fragmentation and DNA oligonucleosomal cleavage, caspases are not activated and caspase inhibitors are ineffective to prevent REGb cell death. In contrast with a number of other types of cell death, the spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide. This unusual mode of cell death that associates necrotic and apoptotic features could provide optimal conditions for triggering a specific immune response.

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.

Protein kinase B (PKB/Akt) is a serine/threonine protein kinase that created serious interest when it was revealed as a mediator of the PI3K pathway. It comprises three isoforms that play both unique and redundant roles. Upon binding to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) generated by PI3K, PKB is phosphorylated by PDK1 at T308. To achieve full kinase activity, PKB needs to be phosphorylated at a second key residue, S473, by members of the PI3K-related kinase family mTORC2 or DNA-PK, depending on the stimulus and the context. Besides, a number of phosphatases and interacting partners have been shown to further modulate its subcellular localization, phosphorylation, and kinase activity. This review aims at illustrating the remarkable complexity in the regulation of PKB signaling downstream of PI3K. Such regulation could be attributed to the specific roles of the PKB isoforms, their expression pattern, subcellular localization, targets, phosphorylation by upstream kinases in a stimulus- and context-dependent manner and by phosphatases, and interaction with binding partners. This allows this key kinase to fulfill physiological functions in numerous processes, including embryonic development, thymocyte development, adipocyte differentiation, glucose homeostasis, and to avoid pathological loss of control such as tumor formation.

Protein kinase B (PKB/Akt) is a serine/threonine protein kinase that created serious interest when it was revealed as a mediator of the PI3K pathway. It comprises three isoforms that play both unique and redundant roles. Upon binding to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) generated by PI3K, PKB is phosphorylated by PDK1 at T308. To achieve full kinase activity, PKB needs to be phosphorylated at a second key residue, S473, by members of the PI3K-related kinase family mTORC2 or DNA-PK, depending on the stimulus and the context. Besides, a number of phosphatases and interacting partners have been shown to further modulate its subcellular localization, phosphorylation, and kinase activity. This review aims at illustrating the remarkable complexity in the regulation of PKB signaling downstream of PI3K. Such regulation could be attributed to the specific roles of the PKB isoforms, their expression pattern, subcellular localization, targets, phosphorylation by upstream kinases in a stimulus- and context-dependent manner and by phosphatases, and interaction with binding partners. This allows this key kinase to fulfill physiological functions in numerous processes, including embryonic development, thymocyte development, adipocyte differentiation, glucose homeostasis, and to avoid pathological loss of control such as tumor formation.

Cellular homeostasis depends upon the strict regulation of responses to external stimuli, such as signalling cascades triggered by nutrients and growth factors, and upon cellular metabolism. One of the major molecules coordinating complex signalling pathways is protein kinase B (PKB), a serine/threonine kinase also known as Akt. The number of substrates known to be phosphorylated by PKB and its interacting partners, as well as our broad understanding of how PKB is implicated in responses to growth factors, metabolic pathways, proliferation, and cell death via apoptosis is constantly increasing. Activated by the insulin/growth factor-phosphatidylinositol 3-kinase (PI3K) cascade, PKB triggers events that promote cell survival and prevent apoptosis. It is also now widely accepted that mitochondria are not just suppliers of ATP, but that they participate in regulatory and signalling events, responding to multiple physiological inputs and genetic stresses, and regulate both cell proliferation and death. Thus, mitochondria are recognized as important players in apoptotic events and it is logical to predict some form of interplay with PKB. In this review, we will summarize mechanisms by which PKB mediates its anti-apoptotic activities in cells and survey recent developments in understanding mitochondrial dynamics and their role during apoptosis.

The highly conserved heat shock proteins (Hsps) accumulate in cells exposed to heat and a variety of other stressful stimuli. Hsps, that function mainly as molecular chaperones, allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked and Hsps induced in response to stress appear to function at key regulatory points in the control of apoptosis. Hsps include anti-apoptotic and pro-apoptotic proteins that interact with a variety of cellular proteins involved in apoptosis. Their expression level can determine the fate of the cell in response to a death stimulus, and apoptosis-inhibitory Hsps, in particular Hsp27 and Hsp70, may participate in carcinogenesis. This review summarizes the apoptosis-regulatory function of Hsps.

The highly conserved heat shock proteins (Hsps) accumulate in cells exposed to heat and a variety of other stressful stimuli. Hsps, that function mainly as molecular chaperones, allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked and Hsps induced in response to stress appear to function at key regulatory points in the control of apoptosis. Hsps include anti-apoptotic and pro-apoptotic proteins that interact with a variety of cellular proteins involved in apoptosis. Their expression level can determine the fate of the cell in response to a death stimulus, and apoptosis-inhibitory Hsps, in particular Hsp27 and Hsp70, may participate in carcinogenesis. This review summarizes the apoptosis-regulatory function of Hsps.

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 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.

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-beta. 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.

HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-alpha). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-kappaB by degrading its main inhibitor, I-kappaBalpha. HSP27 overexpression increases NF-kappaB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and interleukin 1beta. HSP27 does not affect I-kappaBalpha phosphorylation but enhances the degradation of phosphorylated I-kappaBalpha by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-kappaBalpha. A protein complex that includes HSP27, phosphorylated I-kappaBalpha, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-kappaBalpha. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-kappaB activity.

Expression of inducible heat shock protein 70 (HSP70) in tumor cells has been proposed to enhance their immunogenicity. However, HSP70 has also been demonstrated to prevent tumor cell death, a key process for the development of tumor cell immunogenicity. In the present study, we investigated the influence of the HSP70 protein level on PRO colon cancer cell growth and immunogenicity in syngeneic BDIX rats and nude mice. These cells have a basal expression of HSP70 which can be substantially increased by heat shock. When injected subcutaneously in syngeneic animals, PRO cells do not induce any detectable immune response and give rise to progressive, metastatic and lethal tumors. Stable transfection of an anti-sense hsp70 cDNA in PRO cells (PRO-70AS cells) strongly decreased HSP70 expression and sensitized cell-free extracts to cytochrome c/dATP-mediated activation of caspases. Subcutaneous injection of PRO-70AS cells induced tumors that rapidly regressed in syngeneic rats while they grew normally in nude mice. Syngeneic rats injected with PRO-70AS cells became protected against a further challenge with PRO cells. The tumor-specific immune response induced by HSP70-depleted PRO-70AS cells was associated with an increased rate of cell death in vivo. These PRO-70AS cells were also more sensitive to NO-mediated, caspase-dependent, macrophage cytotoxicity in vivo. Altogether, these results indicate that reduced level of HSP70 expression in PRO- colon cancer cells results in the generation of a specific immune response by promoting cell death in vivo.

Stress or heat shock proteins (HSPs) such as HSP27 and HSP70 are expressed in response to a wide variety of physiological and environmental insults including heat, reactive oxygen species or anticancer drugs. Their overexpression allows cells to survive to otherwise lethal conditions. Several different mechanisms may account for the cytoprotective activity of HSP27 and HSP70. First, both proteins are powerful chaperones. Second, both inhibit key effectors of the apoptotic machinery including the apoptosome, the caspase activation complex (both HSP27 and HSP70), and apoptosis inducing factor (only HSP70). Third, they both play a role in the proteasome-mediated degradation of apoptosis-regulatory proteins. HSP27 and HSP70 may participate in oncogenesis, as suggested by the fact that overexpression of heat shock proteins can increase the tumorigenic potential of tumor cells. The down-regulation or selective inhibition of HSP70 might constitute a valuable strategy for the treatment of cancer.

Heat shock protein 70 (HSP70) inhibits apoptosis and thereby increases the survival of cells exposed to a wide range of lethal stimuli. HSP70 has also been shown to increase the tumorigenicity of cancer cells in rodent models. The protective function of this chaperone involves interaction and neutralization of the caspase activator apoptotic protease activation factor-1 and the mitochondrial flavoprotein apoptosis-inducing factor (AIF). In this work, we determined by deletional mutagenesis that a domain of AIF comprised between amino acids 150 and 228 is engaged in a molecular interaction with the substrate-binding domain of HSP70. Computer calculations favored this conclusion. On the basis of this information, we constructed an AIF-derived protein, which is cytosolic, noncytotoxic, yet maintains its capacity to interact with HSP70. This protein, designated ADD70, sensitized different human cancer cells to apoptosis induced by a variety of death stimuli by its capacity to interact with HSP70 and therefore to sequester HSP70. Thus, its chemosensitizing effect was lost in cells in which inducible HSP70 genes had been deleted. These data delineate a novel strategy for the selective neutralization of HSP70.

Heat shock protein 70 (HSP70) can inhibit apoptosis by neutralizing and interacting with apoptosis-inducing factor (AIF), a mitochondrial flavoprotein that translocates upon apoptosis induction to the nucleus, via the cytosol. Here, we show that only members of the HSP70 family interact with AIF. Systematic deletion mapping revealed the existence of three distinct functional regions in the AIF protein: (1) a region between amino acids 150 and 228 that binds HSP70, (2) a domain between residues 367 and 459 that includes a nuclear localization sequence (NLS) and (3) a C-terminal domain beyond residue 567 required for its chromatin-condensing activity. Deletion of the 150-268 domain completely abolished HSP70 binding and facilitated the nuclear import of AIF, resulting in a gain-of-function phenotype with enhanced AIF-mediated chromatin condensation as compared to wild-type AIF. This gain-of-function phenotype was observed in wild-type control cells (which express low but significant levels of HSP70), yet was lost when AIFDelta150-268 was introduced into HSP70 knockout cells, underscoring the functional importance of the AIF-HSP70 interaction. Altogether, our data demonstrate that AIF inhibition by HSP70 involves cytosolic retention of AIF. Moreover, it appears that endogenous HSP70 protein levels are sufficiently elevated to modulate the lethal action of AIF.