PubMed Health⌕ Search

Biomedical subjects

J A Trapani

Publications and source records attributed to J A Trapani.

At least 19 recordsLinked to original sources
PubMed2006

Functional dissociation of DeltaPsim and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2005

Lack of apoptosis of Sézary cells in the circulation following oral bexarotene therapy.

BACKGROUND: Apoptosis of malignant cells has been suggested as an important mechanism of the action of bexarotene in the treatment of cutaneous T-cell lymphoma (CTCL). OBJECTIVES: Our purpose was to examine the in vivo and in vitro responses of patients with Sézary syndrome treated with oral bexarotene and assess them for apoptosis of the Sézary cells. METHODS: Six patients with CTCL with circulating Sézary cells, participating in a clinical trial of oral bexarotene (300 mg m(-2) daily) were included in the study. Peripheral blood from the patients was analysed for in vivo and in vitro apoptosis. RESULTS: None of the six patients demonstrated in vivo apoptosis. In vitro apoptosis of Sézary cells was demonstrated in one patient following exogenous bexarotene. CONCLUSIONS: Apoptosis is not detectable in the circulation of patients with Sézary syndrome treated with bexarotene.

Administration, Oral↗
PubMed2003

Critical role of the transcription factor AP-1 for the constitutive and interferon-induced expression of IFI 16.

IFI 16 is a member of the HIN-200 family of transcriptional regulators that suppress cell growth, modulate the cell cycle and have been linked to cellular differentiation. We hypothesized that the activity of IFI 16 depends on its level of expression and therefore studied the transcriptional activity of the IFI 16 promoter. A discrete sequence within the 5' untranslated region was required for constitutive activity of the promoter and the functional motif within this region was shown to be a consensus AP-1 site. Interestingly, this AP-1 site was also critical for IFN-induced activation of the promoter and consistent with these observations, treatment of cells with IFNgamma resulted in a rapid and robust induction of AP-1 activity that preceded expression of IFI 16. These experiments define the transcriptional mechanisms of IFI 16 gene regulation and provide evidence suggesting that AP-1 activation may be an important event in IFN signaling.

Base Sequence↗
PubMed2002

CTL: Caspases Terminate Life, but that's not the whole story.

The induction of cell death by cytotoxic T-lymphocytes (CTL) or natural killer (NK) cells is one of the main ways by which higher organisms protect themselves from rogue cells, including those infected by a virus, or posing a risk of cancer. Considering the rapidity of viral replication and spread to uninfected cells, CTL and NK are extremely efficient killers. This is at least partly due to the variety of pathways that these cytolytic lymphocytes (CL) can use to ensure the death of a cell. Primarily, CL utilize two independently initiated pathways involving either ligation of death receptors or perforin mediated trafficking of granzyme B to the target cell cytosol to activate a family of death proteases (caspases) in the target cell. The caspases then orchestrate the orderly dismantling of that cell by cleavage of a set of critical substrates. If caspases are inactivated, due either to mutations in proteins that signal their activation or direct inhibition by a viral gene product, CL can utilize a caspase-independent pathway to ensure the death of the target cell. Here we will discuss the mechanisms by which these stellar killers achieve their goal.

Animals↗
PubMed2001

Granzymes: a family of lymphocyte granule serine proteases.

SUMMARY: Granzymes, a family of serine proteases, are expressed exclusively by cytotoxic T lymphocytes and natural killer (NK) cells, components of the immune system that protect higher organisms against viral infection and cellular transformation. Following receptor-mediated conjugate formation between a granzyme-containing cell and an infected or transformed target cell, granzymes enter the target cell via endocytosis and induce apoptosis. Granzyme B is the most powerful pro-apoptotic member of the granzyme family. Like caspases, cysteine proteases that play an important role in apoptosis, it can cleave proteins after acidic residues, especially aspartic acid. Other granzymes may serve additional functions, and some may not induce apoptosis. Granzymes have been well characterized only in human and rodents, and can be grouped into three subfamilies according to substrate specificity: members of the granzyme family that have enzymatic activity similar to the serine protease chymotrypsin are encoded by a gene cluster termed the 'chymase locus'; granzymes with trypsin-like specificities are encoded by the 'tryptase locus'; and a third subfamily cleaves after unbranched hydrophobic residues, especially methionine, and is encoded by the 'Met-ase locus'. All granzymes are synthesized as zymogens and, after clipping of the leader peptide, maximal enzymatic activity is achieved by removal of an amino-terminal dipeptide. They can all be blocked by serine protease inhibitors, and a new group of inhibitors has recently been identified - serpins, some of which are specific for granzymes. Future studies of serpins may bring insights into how cells that synthesize granzymes are protected from inadvertent cell suicide.

Animals↗
PubMed2001

Redirecting mouse CTL against colon carcinoma: superior signaling efficacy of single-chain variable domain chimeras containing TCR-zeta vs Fc epsilon RI-gamma.

The structurally related TCR-zeta and Fc receptor for IgE (Fc epsilon RI)-gamma are critical signaling components of the TCR and Fc epsilon RI, respectively. Although chimeric Ab receptors containing zeta and gamma signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the zeta and gamma subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-zeta and scFv-gamma receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-zeta chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-gamma receptors. T cells expressing scFv-zeta chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the zeta signaling pathway.

3T3 Cells↗
PubMed2001

Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16.

Members of the interferon-induced class of nuclear factors possess a putative CcN motif, comparable with that within proteins such as the simian virus 40 large tumour antigen (T-ag), which confers phosphorylation-mediated regulation of nuclear-localization sequence (NLS)-dependent nuclear import. Here we examine the functionality of the interferon-induced factor 16 (IFI 16) CcN motif, demonstrating its ability to target a heterologous protein to the nucleus, and to be phosphorylated specifically by the CcN-motif-phosphorylating protein kinase CK2 (CK2). The IFI 16 NLS, however, has novel properties, conferring ATP-dependent nuclear import completely independent of cytosolic factors, as well as binding to nuclear components. The IFI 16 NLS is not recognized with high affinity by the NLS-binding importin heterodimer, and transport mediated by it is insensitive to non-hydrolysable GTP analogues. The IFI 16 NLS thus mediates nuclear import through a pathway completely distinct from that of conventional NLSs, such as that of T-ag, but intriguingly resembling that of the NLS of the HIV-1 transactivator protein Tat. Since the IFI 16 CK2 site enhances nuclear import through facilitating binding to nuclear components, this represents a novel mechanism by which the site regulates nuclear-protein import, and constitutes a difference between the IFI 16 and Tat NLSs that may be of importance in the immune response.

Active Transport, Cell Nucleus↗
PubMed2001

Granzyme A and B-deficient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo anti-tumor capacity.

Recent studies have demonstrated that granzymes A and B make an important contribution to the clearance of the orthopoxvirus ectromelia, and in graft versus host disease. To test whether granzymes are generally necessary for lymphocyte-mediated cytotoxicity in vivo, we assessed the cytotoxic capacity of granzyme A and/or B-deficient lymphocytes in several perforin-dependent settings. Splenocytes and allogeneic CTL of granzyme A and/or B-deficient mice were defective for induction of DNA fragmentation, but induced significant membrane damage and target cell death. These results correlated well with the behavior of granzyme A/B-deficient CTL and NK cells in three different perforin-dependent tumor models. In a classical assay of NK cell-mediated rejection, granzyme A and/or B-deficient mice inoculated with RMA-S cells were as susceptible to tumor as wild-type mice. Perforin-deficient mice were also considerably more susceptible to tumor initiation by methylcholanthrene than granzyme A and/or B-deficient mice. Furthermore, rejection of the K1735-melanoma expressing MHC class I and II molecules was mediated by adoptively transferred H-2b anti-k CTL from immunized granzyme A and/or B-deficient mice. In summary, these data suggest that granzymes A and B are not critical for most anti-tumor effector functions of NK cells and CTL that are perforin mediated.

Animals↗
PubMed2001

Lymphocyte-mediated immunosurveillance of epithelial cancers?

The validity and importance of the immunosurveillance of tumors have been debated ever since Burnet and Thomas outlined several key concepts that underpin our understanding of tumor immunity. With the development of gene-targeting technology, it has been shown that interferon gamma (IFN-gamma) and perforin--key effector molecules of both the innate and adaptive immune systems--contribute to the prevention of of tumors in mice. These findings have rejuvenated interest in the immunosurveillance of tumors, and recent data have provided evidence that the immune system influences the development of some spontaneous epithelial malignancies.

Animals↗
PubMed2001

A fresh look at tumor immunosurveillance and immunotherapy.

Despite major advances in our understanding of adaptive immunity and dendritic cells, consistent and durable responses to cancer vaccines remain elusive and active immunotherapy is still not an established treatment modality. The key to developing an effective anti-tumor response is understanding why, initially, the immune system is unable to detect transformed cells and is subsequently tolerant of tumor growth and metastasis. Ineffective antigen presentation limits the adaptive immune response; however, we are now learning that the host's innate immune system may first fail to recognize the tumor as posing a danger. Recent descriptions of stress-induced ligands on tumor cells recognized by innate effector cells, new subsets of T cells that regulate tumor tolerance and the development of spontaneous tumors in mice that lack immune effector molecules, beckon a reflection on our current perspectives on the interaction of transformed cells with the immune system and offer new hope of stimulating therapeutic immunity to cancer.

Animals↗
PubMed2001

Perforin-independent expression of granzyme B and proteinase inhibitor 9 in human testis and placenta suggests a role for granzyme B-mediated proteolysis in reproduction.

Granzyme B (graB) plays a pivotal role in cytotoxic lymphocyte granule-mediated apoptosis through cleavage of intracellular proteins in target cells. Proteinase inhibitor-9 (PI-9) is a potent inhibitor of graB and is highly expressed in cytotoxic lymphocytes. Here, we show by immunohistochemistry that PI-9 is also abundantly expressed in human testicular Sertoli cells and placental syncytial trophoblasts. Postulating that PI-9 protects these tissues from graB-producing auto- or allo-reactive cytotoxic lymphocytes, we also stained sections for graB. Unexpectedly, graB was observed in non-cytotoxic cells in both tissues. In the adult human testis, graB was present in spermatogenic cells within the seminiferous tubule, and this was verified by in-situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical analysis of term placentae demonstrated graB in syncytial trophoblasts, and this was confirmed by RT-PCR on primary trophoblasts from term placenta. Perforin, which is co-produced with graB by activated cytotoxic lymphocytes and is required for graB release into the target cell, was not detected in either testis or placenta. We postulate that, in these organs, graB has a perforin-independent role, involving hydrolysis of extracellular matrix components. In the testis, graB may facilitate migration of developing germ cells, while in the placenta, it may contribute to extracellular matrix remodelling during parturition.

Animals↗
PubMed2001

Unlocking the secrets of cytotoxic granule proteins.

Cytotoxic lymphocytes largely comprise CD8(+) cytotoxic T cells and natural killer cells and form the major defense of higher organisms against virus-infected and transformed cells. A key function of cytotoxic lymphocytes is to detect and eliminate potentially harmful cells by inducing them to undergo apoptosis. This is achieved through two principal pathways, both of which require direct but transient contact between the killer cell and its target. The first, involving ligation of TNF receptor-like molecules such as Fas/CD95 by their cognate ligands, results in mobilization of conventional, programmed cell-death pathways centered on activation of pro-apoptotic caspases. This review concentrates on the second pathway, in which the toxic contents of secretory vesicles of the cytotoxic lymphocyte are secreted toward the target cell, and some toxins penetrate into the target cell cytoplasm and nucleus. In addition to invoking a powerful stimulus to caspase activation, this "granule-exocytosis mechanism" provides a variety of additional strategies for overcoming inhibitors of the caspase cascade that may be elaborated by viruses. The key molecular players in this process are the pore-forming protein perforin and a family of granule-bound serine proteases or granzymes. The molecular functions of perforin and granzymes are under intense investigation in many laboratories including our own, and recent advances will be discussed. In addition, this review discusses the evidence pointing to the importance of perforin and granzyme function in pathophysiological situations as diverse as infection with intracellular pathogens, graft versus host disease, susceptibility to transplantable and spontaneous malignancies, lymphoid homeostasis, and the tendency to auto-immune diseases.

Animals↗
PubMed2001

Mechanisms of interferon mediated anti-viral resistance.

Interferons (IFNs) are an important part of immune responses and are believed to protect the host from viral and bacterial pathogens as well as having a role in rejection of malignancies. The well-known anti-viral and cytostatic properties of IFNs have led to the clinical use of these proteins to treat some cancers and viral infections. Extensive research has begun to unravel much of the molecular basis for the biological effects of IFNs, and this information could now be used as a foundation for the development of novel therapeutic strategies that avoid some of the acknowledged shortcomings of cytokine therapies. This review explains the current model of IFN action, during viral infections and the potential for well-established and emerging groups of IFN inducible genes as therapeutic targets is highlighted.

Animals↗
PubMed2000

Filamin (280-kDa actin-binding protein) is a caspase substrate and is also cleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis.

We used yeast two-hybrid screening to identify the cytoskeletal protein filamin as a ligand for the proapoptotic protease granzyme B, produced by cytotoxic T lymphocytes. Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it was also cleaved in a caspase-dependent manner following the ligation of Fas receptors. A similar pattern of filamin cleavage to polypeptides of approximately 110 and 95 kDa was observed in Jurkat cells killed by either mechanism. However, filamin cleavage in response to granzyme B was not inhibited by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations that abolished DNA fragmentation. Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following ligation of Fas receptors, coincident with the morphological changes of apoptosis. Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic survival and (51)Cr release assays, whereas death from multiple other stimuli was not affected by filamin deficiency. Thus, filamin is a functionally important substrate for granzyme B, as its cleavage may account at least partly for caspase-independent cell death mediated by the granzyme.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Functional interaction between p53 and the interferon-inducible nucleoprotein IFI 16.

Interferons are important in regulating cell growth and differentiation, immune function and initiating anti-viral responses. While the pleotrophic actions of interferons have been well documented, the molecular mechanisms underpinning their biological effects have not been fully characterized. IFI 16 is a member of the interferon-inducible HIN-200 family of nuclear proteins, which we have recently shown can function as a potent transcriptional repressor. A murine member of the HIN-200 family, p202, can indirectly interact with p53 via the p53 binding protein (p53bp) and inhibit p53-mediated transcriptional activation. The binding activity of p202 to p53bp was shown to require the conserved MFHATVAT motif present in all 200 amino acid repeat regions of HIN-200 proteins. Given that IFI 16 contains two MFHATVAT motifs, we sought to determine whether IFI 16 may form a complex with p53 and if so to ascertain the functional significance of this interaction. We demonstrate that IFI 16 can directly bind to the C-terminal region of p53 and augment p53-mediated transcriptional activation without altering the steady state levels of p53. Thus, in addition to its ability to directly regulate gene expression, IFI 16 can also modulate the transcription function of other cellular transcription factors. These findings demonstrate a possible link between gene induction following interferon stimulation and p53-mediated cellular events.

Amino Acid Motifs↗
PubMed2000

Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.

The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Alkyl and Aryl Transferases↗