Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane β- barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM- and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22- and mtHsp70-dependent import pathway

Granzyme A, which causes single‐stranded DNA damage, targets the double‐strand break repair protein Ku70

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects—facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70

miR-200 Enhances Mouse Breast Cancer Cell Colonization to Form Distant Metastases

BACKGROUND: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells. METHODOLOGY/PRINCIPAL FINDINGS: miRNA expression was analyzed by miRNA microarray and quantitative RT-PCR in isogenic mouse breast cancer cells with distinct metastatic capabilities. 4T1 cells that form macroscopic metastases had elevated expression of miR-200 family miRNAs compared to related cells that invade distant tissues, but are unable to colonize. Moreover, over-expressing miR-200 in 4TO7 cells enabled them to metastasize to lung and liver. These findings are surprising since the miR-200 family was previously shown to promote epithelial characteristics by inhibiting the transcriptional repressor Zeb2 and thereby enhancing E-cadherin expression. We confirmed these findings in these cells. The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells. CONCLUSIONS/SIGNIFICANCE: Expression of miR-200, which promotes a mesenchymal to epithelial cell transition (MET) by inhibiting Zeb2 expression, unexpectedly enhances macroscopic metastases in mouse breast cancer cell lines. These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET. Therefore the epithelial nature of a tumor does not predict metastatic outcome

ROS signaling during granzyme B-mediated apoptosis

Reactive oxygen species (ROS) are involved in cell signaling, aging, and death and play a role in carcinogenesis. However, whether ROS are bystanders or active effectors of apoptosis was unclear until recently. New evidence shows that the killer lymphocyte protease granzyme B activates a conserved biochemical pathway centered on respiratory chain disruption to trigger mitocentric ROS-dependent apoptosis

INTERACTIONS CELLULAIRES DANS LES LYMPHOMES T CUTANES ET MECANISMES D'ECHAPPEMENT TUMORAL

LES LYMPHOMES T CUTANES (CTCL) DONT LE MYCOSIS FONGOIDE (MF) SONT DES DESORDRES LYMPHOPROLIFERATIFS DE CELLULES T MONOCLONALES DE PHENOTYPE CD4 + CD45RO + CD8 . NOUS AVONS CHERCHE A ETUDIER LES INTERACTIONS CELLULAIRES DANS LES LYMPHOMES T CUTANES ET CARACTERISER LES MECANISMES D'ECHAPPEMENT A LA REPONSE IMMUNITAIRE. MALGRE UNE EXPRESSION DU RECEPTEUR POUR LE TNF (P55 ET P75), DE FAS ET FASL, LES CELLULES TUMORALES SONT RESISTANTES A L'ACTION DU TNF ET L'ACTIVITE CYTOTOXIQUE DES CLONES TC5 ET TC7 EXPRIMANT FASL PASSE PAR LA VOIE PERFORINE/GRANZYME ET PROBABLEMENT PAR TRAIL. DES ETUDES PAR RT-PCR DE L'EXPRESSION DES MESSAGERS CODANT POUR LES CYTOKINES ONT MONTRE QUE LES CLONES TC7 ET TC5 ONT UN PROFIL TYPE TH-1 (IL2, IFN ) ET LE GM-CSF ALORS QUE LES CELLULES TUMORALES ONT UN PROFIL TH-2 (IL4, IL6, IL10) ET LE TGF. CES RESULTATS CONFIRMENT, D'UNE PART LE CARACTERE DE CTL ANTI-TUMORAL DES CLONES TC5 ET TC7 ET D'AUTRE PART, DEMONTRENT QUE LE PROFIL CYTOKINIQUE DES CELLULES TUMORALES PEUT DIMINUER LA REPONSE ANTI-CANCEREUSE, FAVORISANT L'ECHAPPEMENT DE LA TUMEUR A LA REPONSE IMMUNITAIRE. NOUS AVONS AUSSI ISOLE UN TROISIEME CLONE ANTI-TUMORAL RESTREINT PAR HLA-A2. CE CLONE CD8 DEN-1 A REARRANGE LES SEGMENTS V13.2-J2.5 COMME LA LIGNEE TUMORALE. DEN-1 EXPRIME LES KIRS P58A, P58B, P70 ET CD94. CES RESULTATS DEMONTRENT POUR LA PREMIERE FOIS QUE L'EXPRESSION DE KIRS PEUT CONDUIRE A L'INHIBITION DE LA REPONSE T SPECIFIQUE DANS LE LYMPHOME T CUTANE. UNE TROISIEME LIGNEE TUMORALE COUL3, QUI PRESENTE UNE AUTORECONNAISSANCE ACCOMPAGNEE D'UNE AUTOPROLIFERATION A ETE ISOLEE. CETTE AUTORECONNAISSANCE N'ETAIT PAS ACCOMPAGNEE D'AUTOCYTOTOXICITE MALGRE UN TCR FONCTIONNEL. LE FAIT QUE LE CLONE COU-L3 PRESENTE UNE AUTOREACTIVITE ENTRAINANT L'AUTOACTIVATION ET LA PROLIFERATION CELLULAIRE, SUPPOSE L'EXISTENCE D'UNE BOUCLE POSITIVE AUTOCRINE D'ACTIVATION FAVORISANT LA TUMEUR.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis

none3noneMartinvalet, Denis; Zhu, Pengcheng; Lieberman, Judy*Martinvalet, Denis; Zhu, Pengcheng; Lieberman, Jud

Granzymes and cell death

Granzymes are cell death-inducing serine proteases released from cytotoxic granules of cytotoxic T lymphocytes and natural killer cells during granule exocytosis in response to viral infection or against transformed cells marked for elimination. A critical cofactor for the granule exocytosis pathway is perforin, which mediates the entry of granzymes into target cells, where they cleave specific substrates that initiate DNA fragmentation and apoptosis. One of the biggest challenges in studying the biology of granzymes has been the functional redundancy of granzymes in animal models making an in vitro experimental system essential. This chapter discusses methods to study granzyme function in vitro under physiologically relevant experimental conditions

Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability

Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies