Involvement of CASP9 (caspase 9) in IGF2R/CI-MPR endosomal transport.

Recently, we reported that increased expression of CASP9 pro-domain, at the endosomal membrane in response to HSP90 inhibition, mediates a cell-protective effect that does not involve CASP9 apoptotic activity. We report here that a non-apoptotic activity of endosomal membrane CASP9 facilitates the retrograde transport of IGF2R/CI-MPR from the endosomes to the trans-Golgi network, indicating the involvement of CASP9 in endosomal sorting and lysosomal biogenesis. CASP9-deficient cells demonstrate the missorting of CTSD (cathepsin D) and other acid hydrolases, accumulation of late endosomes, and reduced degradation of bafilomycin A-sensitive proteins.

HSP90 inhibition targets autophagy and induces a CASP9-dependent resistance mechanism in NSCLC.

Macroautophagy/autophagy has emerged as a resistance mechanism to anticancer drug treatments that induce metabolic stress. Certain tumors, including a subset of KRAS-mutant NSCLCs have been shown to be addicted to autophagy, and potentially vulnerable to autophagy inhibition. Currently, autophagy inhibition is being tested in the clinic as a therapeutic component for tumors that utilize this degradation process as a drug resistance mechanism.

A Complex between Atg7 and Caspase-9: A NOVEL MECHANISM OF CROSS-REGULATION BETWEEN AUTOPHAGY AND APOPTOSIS.

Several cross-talk mechanisms between autophagy and apoptosis have been identified, in which certain co-regulators are shared, allowing the same protein to participate in these opposing processes. Our studies suggest that caspase-9 is a novel co-regulator of apoptosis and autophagy and that its caspase catalytic activity is dispensable for its autophagic role. We provide evidence that caspase-9 facilitates the early events leading to autophagosome formation; that it forms a complex with Atg7; that Atg7 is not a direct substrate for caspase-9 proteolytic activity; and that, depending on the cellular context, Atg7 represses the apoptotic capability of caspase-9, whereas the latter enhances the Atg7-mediated formation of light chain 3-II.

Interaction between Her2 and Beclin-1 proteins underlies a new mechanism of reciprocal regulation.

Beclin-1 is a key regulator of autophagy that functions in the context of two phase-specific complexes in the initiation and maturation of autophagosomes. Its known interacting proteins include autophagy effectors, Bcl-2 family members, and organelle membrane anchor proteins. Here we report a newly identified interaction between Beclin-1 and the protein tyrosine kinase receptor Her2.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.

Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis.

Apoptotic defects endow tumor cells with survival advantages. Such defects allow the cellular stress response to take the path of cytoprotective autophagy, which either precedes or effectively blocks an apoptotic cascade. Inhibition of the cytoprotective autophagic response shifts the cells toward apoptosis, by interfering with an underlying molecular mechanism of cytoprotection.

Regulation of mitochondrial apoptotic events by p53-mediated disruption of complexes between antiapoptotic Bcl-2 members and Bim.

Multiple mechanisms have been proposed for the mitochondrial function of p53 that are either dependent on or independent of its transcriptional activity. However, none of these mechanisms involves Bim functioning downstream of p53 mitochondrial translocation. Utilizing a p53 nuclear localization signal mutant, whose nuclear import is completely abrogated, we demonstrate that its apoptotic activity at the outer mitochondrial membrane, which involves conformational changes in Bax and Bak, is mediated by Bim.

Deregulation of mitochondrial membrane potential by mitochondrial insertion of granzyme B and direct Hax-1 cleavage.

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion.

Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2).

Human CD4(+)CD25(high)FOXP3(+) T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death.

Enhancement of tumor-TRAIL susceptibility by modulation of autophagy.

Autophagy has recently been recognized as an important cellular response to stress. However, the prospect of manipulating the autophagic process for the enhancement of cancer therapy remains unresolved. This lack of resolution stems from the current controversy regarding the fundamental function of autophagy in tumor stress response: Does it have a positive or negative impact on tumor survival capability? Our studies were designed to investigate the role of autophagy in the response to TRAIL of tumor cells with various apoptotic defects.

Expression of X-linked inhibitor-of-apoptosis protein in hepatocellular carcinoma promotes metastasis and tumor recurrence.

Unlabelled: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Despite significantly improved diagnosis and treatment in recent years, the long-term therapeutic effect is compromised by the frequent recurrence and metastasis, of which the molecular mechanisms are not fully understood. Our initial studies in established HCC cell lines with different metastatic capabilities indicated a correlation of metastasis with the resistance to apoptosis and therefore the ability to survive in stressed conditions.

Involvement of protective autophagy in TRAIL resistance of apoptosis-defective tumor cells.

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy.

Functional linkage between NOXA and Bim in mitochondrial apoptotic events.

NOXA is a BH3-only protein whose expression is induced by certain p53-depenent or independent apoptotic stimuli. Both NOXA and Bim are avid binders of Mcl-1, but a functional linkage between these BH3-only proteins has not yet been reported. In this study, we demonstrate that Mcl-1 binding of endogenously induced NOXA interferes with the ability of Mcl-1 to efficiently sequester endogenous Bim, as Bim is displaced from its complex with Mcl-1.

Cytolytic cells induce HMGB1 release from melanoma cell lines.

High mobility group box 1 (HMGB1) is one of the recently defined damage-associated molecular pattern molecules, passively released from necrotic cells and secreted by activated macrophage/monocytes. Whether cytolytic cells induce HMGB1 release from tumor cells is not known. We developed a highly sensitive method for detecting intracellular HMGB1 in tumor cells, allowing analysis of the type of cell death and in particular, necrosis.

Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis.

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1.

Disruption of Mcl-1.Bim complex in granzyme B-mediated mitochondrial apoptosis.

Recently, we reported the identification of a novel mitochondrial apoptotic pathway for granzyme B (GrB). The newly identified GrB-mediated mitochondrial cascade was initiated by the cleavage and subsequent degradation of Mcl-1, resulting in the release of mitochondrial Bim from Mcl-1 sequestration. To investigate the biological significance of Mcl-1 cleavage by GrB, we mapped the major GrB cleavage sites and evaluated the apoptotic potential of the cleavage products.

Differential involvement of Bax and Bak in TRAIL-mediated apoptosis of leukemic T cells.

TRAIL-induced apoptosis has been considered a promising therapeutic approach for tumors that are resistant to chemotherapy, which is usually mediated via mitochondrial apoptotic cascades. Recent studies have shown that in certain cancer cells, TRAIL-mediated apoptosis is also dependent on mitochondrial involvement, suggesting that similar mechanisms of resistance to chemotherapy might be implicated in the resistance of tumor cells to TRAIL. We have used TRAIL-resistant leukemic cells that are deficient in both Bax and Bak to determine the roles of these Bcl-2 members in TRAIL-mediated apoptosis.

Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome C release.

Release of cytochrome c from the mitochondrial intermembrane space is critical to apoptosis induced by a variety of death stimuli. Bid is a BH3-only prodeath Bcl-2 family protein that can potently activate this efflux. In the current study, we investigated the mitochondrial localization of Bid and its interactions with mitochondrial phospholipids, focusing on their relationships with Bid-induced cytochrome c release.

Degradation of Mcl-1 by granzyme B: implications for Bim-mediated mitochondrial apoptotic events.

Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim.

A novel apoptotic pathway as defined by lectin cellular initiation.

In this study of lectin-induced apoptosis we found that wheat germ agglutinin (WGA) initiated an accelerated type of programmed cell death developing after only 30 min of incubation with tumor cells. To analyze possible mechanisms, studies were focused using the WGA lectin whose carbohydrate specificity is well defined. We found that WGA could induce apoptosis by binding to either N-acetylneuraminic acid or N-acetylglucosamine (GlcNAc) on the cell surface of normal and malignant cells.

Identification of a synovial fibroblast-specific protein transduction domain for delivery of apoptotic agents to hyperplastic synovium.

Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pathologies associated with rheumatoid arthritis. To develop an approach for efficient delivery of proteins or agents to synovium to induce targeted apoptosis of hyperplastic synovial tissue, we have screened an M13 peptide phage display library for synovial-specific transduction peptides. We identified a novel synovial-targeted transduction peptide, HAP-1, which is able to facilitate specific internalization of protein complexes into human and rabbit synovial cells in culture and rabbit synovial lining in vivo.

Expression of zeta in T cells prior to interleukin-2 therapy as a predictor of response and survival in patients with ovarian carcinoma.

Expression levels of T-cell receptor (TcR)-associated zeta chain were reported to reflect functional competence of T lymphocytes in patients with cancer. This retrospective study was performed to evaluate zeta chain expression in circulating T cells obtained from clinical responders and nonresponders among 19 patients with advanced ovarian carcinoma treated with intraperitoneal interleukin-2 (IL-2) biotherapy. Banked lymphocytes, which were collected from the patients who participated in a phase I clinical trial performed between 1987 and 1990, were used for quantitative flow cytometry to measure zeta-chain expression in T lymphocytes prior to and at the end of therapy.

Granzyme B-mediated degradation of T-cell receptor zeta chain.

We recently reported that the T-cell receptor (TCR)-zeta chain is cleaved by caspase-3 and -7 in apoptotic T lymphocytes or in a cell-free system. We report here that the zeta chain is also a direct substrate for granzyme B (GrB) proteolytic activity. Loss in expression of TCR-zeta was observed in Jurkat T leukemic cells treated by a combination of GrB and a replication-deficient adenovirus.