TPCK targets elements of mitotic spindle and induces cell cycle arrest in prometaphase.

The serine protease inhibitor N-alpha-tosyl-epsilon-phenylalanyl chloromethyl ketone (TPCK) has been long used in studies of cellular processes including apoptosis. Depending on the experimental conditions, TPCK either induces or inhibits changes associated with apoptosis but there has been little progress in identifying the relevant targets for TPCK. Our group recently showed that the largest subunit of the RNA polymerase II is one of the intracellular targets of TPCK. The complex effects of TPCK on apoptosis, however, suggested the existence of additional apoptosis-relevant targets in cells. Using our unique polyclonal anti-tosyl antibody, here we report the identification of the mitotic spindle as another intracellular target for TPCK. We also provide data that TPCK-mediated labeling of the mitotic spindle correlates with cell cycle arrest in prometaphase.

TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells.

N-alpha-Tosyl-L: -phenylalanyl chloromethyl ketone (TPCK) is an affinity label for chymotrypsin-like proteases and has been extensively used as an experimental tool in apoptosis research to probe the role of proteases in cell death. While TPCK blocks some apoptotic changes and induces others, the cellular target or targets for TPCK have not been identified. Here we investigated for the first time the cellular targets for TPCK using a polyclonal anti-tosyl antibody. We have found that TPCK rapidly and irreversibly labels numerous intracellular proteins and have identified one as RPB1, the largest subunit of RNA polymerase II. We show that TPCK inhibits DNA binding by RNA polymerase and that TPCK inhibits transcription. Inhibition of transcription is known to induce apoptosis and while TPCK may trigger death through interaction with multiple targets, our data suggests that the pro-apoptotic effects of TPCK may be explained in part by the inhibition of RNA polymerase II activity.

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis.

Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis.

Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family.

Inhibitor of apoptosis protein (IAP)-like protein-1 (ILP-1) (also known as X-linked IAP [XIAP] and mammalian IAP homolog A [MIHA]) is a potent inhibitor of apoptosis and exerts its effects, at least in part, by the direct association with and inhibition of specific caspases. Here, we describe the molecular cloning and characterization of a human gene related to ILP-1, termed ILP-2. Despite high homology to ILP-1, ILP-2 is encoded by a distinct gene, which in normal tissues is expressed solely in testis. In contrast to ILP-1, overexpression of ILP-2 had no protective effect on apoptosis mediated by Fas (also known as CD95) or tumor necrosis factor. However, ILP-2 potently inhibited apoptosis induced by overexpression of Bax or by coexpression of caspase 9 with Apaf-1, and preincubation of cytosolic extracts with ILP-2 abrogated caspase activation in vitro. A processed form of caspase 9 could be coprecipitated with ILP-2 from cells, suggesting a physical interaction between ILP-2 and caspase 9. Thus, ILP-2 is a novel IAP family member with restricted specificity for caspase 9.

An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells.

Apoptosis was induced in THP.1 cells, a human monocytic tumour cell line, by diverse stimuli including cycloheximide, thapsigargin, etoposide and staurosporine. Induction of apoptosis by all these stimuli, except etoposide, was enhanced in the presence of the trypsin-like protease inhibitor, N alpha-tosyl-L-lysinyl chloromethyl ketone (TLCK). Induction of apoptosis, assessed by morphological, flow cytometric and biochemical criteria, including proteolysis of poly(ADP-ribose) polymerase and cleavage of DNA to large kilobasepair fragments, was completely abrogated when cells were pretreated with an ICE-like protease inhibitor, Z-Val-Ala-Asp.fluoromethylketone. This suggested that an ICE homologue was a common mediator of apoptosis in THP.1 cells.

An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes.

Apoptosis was induced in thymocytes using diverse stimuli in order to identify events within a common apoptotic pathway. Benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone (Z-VAD.FMK), an interleukin-1 beta-converting enzyme (ICE)-like protease inhibitor, inhibited apoptosis assessed by flow cytometry, proteolysis of poly (ADP)-ribose polymerase of DNA to both large kilobase pair fragments (30-50 and 200-300 kbp) and to nucleosomal fragments. Z-VAD.FMK also blocked all the classical ultrastructural features of apoptosis including chromatin condensation to one pole of the nucleus, nucleolar disintegration and cytoplasmic vacuolation. These results suggest the involvement of an ICE-like protease as a common mediator of apoptosis in thymocytes.

A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the inhibitors N-tosyl-L-lysyl chloromethylketone (TLCK) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) have very different effects on apoptosis. TLCK inhibits apoptosis induced by diverse stimuli at an early stage prior to both DNA fragmentation and cytoplasmic changes. We show that the TLCK-sensitive target is pre-existing and not synthesized in response to apoptotic stimuli. The contrasting effects of TLCK and TPCK support the hypothesis that the TLCK target is a trypsin-like protease which is a common effector of thymocyte apoptosis.

Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2.

An assay for the autoubiquitination activity of the E3 ligase HDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used to measure the activity of other E3s and may be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

Dexamethasone and etoposide induce apoptosis in rat thymocytes from different phases of the cell cycle.

Dexamethasone and etoposide both induce apoptosis in immature rat thymocytes. We investigated the dependence of apoptosis on the phase of the cell cycle after incubation with these drugs. Cell cycle progression was followed by a combination of pulse labelling with 5-bromo-2'-deoxyuridine (BrdU), labelling fixed cells with an anti-BrdU antibody and flow cytometry. Dexamethasone had little effect on the cell cycle progression of proliferating thymocytes, while etoposide caused cell cycle arrest. Normal and apoptotic thymocytes were separated by centrifugation on discontinuous Percoll gradients into four fractions (F1-F4). It was found that both dexamethasone and etoposide induced apoptosis in cells in G0/G1 and G2/M of the cell cycle, whereas only etoposide induced apoptosis of cells in S phase. These results demonstrated that dexamethasone induced apoptosis in quiescent cells while only etoposide could induce apoptosis in cells from the proliferative compartment. Following treatment of thymocytes with etoposide, some of the proliferating thymocytes (F1) were converted to cells with intermediate size and density (F3). We have recently identified these cells as a population of preapoptotic thymocytes, at an early stage of apoptosis. These cells then further progressed to fully apoptotic cells (F4). These data support the hypothesis that normal thymocytes (F1) became apoptotic (F4) via an intermediate population (F3).

DNA degradation and proteolysis in thymocyte apoptosis.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone (Z-VAD.FMK) inhibit apoptosis. N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) has a very different effect, inducing the early morphological and biochemical changes associated with apoptosis. TLCK inhibits trypsin-like proteases whilst Z-VAD.FMK inhibits interleukin-1 beta-converting enzyme (ICE)-like proteases; this and the contrasting effects of TPCK support the hypothesis that thymocyte apoptosis involves a hierarchy of proteases which act at different stages of the process.

Selective repression of the oncogene cyclin D1 by the tumor suppressor miR-206 in cancers.

MicroRNAs (miRNAs) are deregulated in cancer and have been shown to exhibit both oncogenic and tumor suppressive functions. Although the functional effects of several miRNAs have been elucidated, those of many remain to be discovered. In silico analysis identified microRNA-206 (miR-206) binding sites in the 3'-untranslated regions (3'-UTR) of both the mouse and human CCND1 gene. Cyclin D1 is a recognized oncogene involved in direct phosphorylation of the retinoblastoma (Rb) protein and promoting cell cycle transition from G1 to S. miR-206 specifically binds to the CCND1 3'-UTR and mediates reduction of both cyclin D1 protein and mRNA. Expression of miR-206 induced a G1 arrest and a decrease in cell proliferation in breast cancer cells. Ectopic expression of miRNA-resistant cyclin D1 was able to reverse the miR-206-induced decrease in cell proliferation. Therefore, we identified miR-206 as an activator of cell cycle arrest resulting in a decrease in cell proliferation that is dependent on the inhibition of cyclin D1. Interestingly, prostatic cancer (PCa) cells express low levels of miR-206 resulting in deregulated cyclin D1 expression compared with non-transformed primary prostatic epithelial cells (PrEC). Finally, we demonstrate that cyclin D1 is regulated by miR-206 in PrEC but not in PCa cells and this is due to the absence of a CCND1 3'-UTR in these cells. This suggests that miR-206-based anti-cyclin D1 targeted therapy would be beneficial in cancers where cyclin D1 is overexpressed and contains a 3'-UTR.

Caspases as therapeutic targets.

The development of small molecules to modulate caspase activity offers a novel therapeutic strategy in the treatment of apoptosis-related and inflammatory diseases. Caspases are key mediators of apoptosis and inflammation; deregulation of their activation or expression can lead to the development of conditions such as neurodegenerative and autoinflammatory disorders. This review details the different caspase-associated disorders while focusing on caspase-1 inhibition as a potential therapeutic strategy. Problems facing the development of effective and safe caspase therapeutics will also be addressed.

Oncogene-dependent apoptosis in extracts from drug-resistant cells.

Many genotoxic agents kill tumor cells by inducing apoptosis; hence, mutations that suppress apoptosis produce resistance to chemotherapy. Although directly activating the apoptotic machinery may bypass these mutations, how to achieve this activation in cancer cells selectively is not clear. In this study, we show that the drug-resistant 293 cell line is unable to activate components of the apoptotic machinery-the ICE-like proteases (caspases)-following treatment with an anticancer drug. Remarkably, extracts from untreated cells spontaneously activate caspases and induce apoptosis in a cell-free system, indicating that drug-resistant cells have not only the apoptotic machinery but also its activator. Comparing extracts from cells with defined genetic differences, we show that this activator is generated by the adenovirus E1A oncogene and is absent from normal cells. We provide preliminary characterization of this oncogene generated activity (OGA) and show that partially purified OGA activates caspases when added to extracts from untransformed cells. We suggest that agents that link OGA to caspases in cells would kill tumor cells otherwise resistant to conventional cancer therapy. As this killing relies on an activity generated by an oncogene, the effect of these agents should be selective for transformed cells.

Oncogene-dependent apoptosis is mediated by caspase-9.

Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as reflected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked drug-induced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria.