Chimeric caspase molecules with potent cell killing activity in apoptosis-resistant cells.

Cellular defects which prevent apoptotic cell death can result in the generation of hyperproliferative disorders and can prevent the effective treatment of such diseases. The majority of cellular defects which result in apoptosis resistance lie upstream of caspase activation. We have described chimeric caspase molecules consisting of the prodomain of caspase-2 fused to the amino terminus of caspase-3, and which are tagged at the carboxyl terminus with green fluorescent protein (GFP) to allow direct visualisation of transfected cells. Here we show that these chimeric caspase molecules possess potent, rapid cell-killing activity in cell lines which display a range of defects resulting in apoptosis resistance.

Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD.

RAIDD, a caspase recruitment domain (CARD) containing molecule, interacts with procaspase-2 in a CARD-dependent manner. This interaction has been suggested to mediate the recruitment of caspase-2 to the tumour necrosis factor receptor 1 (TNFR1). In this paper we have studied the subcellular localization of RAIDD and its interaction with caspase-2. We demonstrate that endogenous RAIDD is mostly localized in the cytoplasm and to some extent in the nucleus. RAIDD localization is not affected by TNF-treatment of HeLa cells, but in cells ectopically expressing caspase-2, a fraction of RAIDD is recruited to the nucleus. In transfected cells, coexpression of RAIDD and caspase-2 leads to CARD-dependent colocalization of the two proteins to discrete subcellular structures. We further show that overexpression of the RAIDD-CARD results in the formation of filamentous structures due to CARD-mediated oligomerization. These structures were similar to death effector filaments (DEFs) formed by FADD and FLICE death effector domains (DEDs), and partially colocalized with DEFs. Our results suggest that similar to the DED, the RAIDD-CARD has the ability to form higher order complexes, believed to be important in apoptotic execution. We also present evidence that RAIDD-CARD oligomerization may be regulated by intramolecular folding of the RAIDD molecule.

Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum).

We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N-linked glycosylation sites, a potential N-terminal signal peptide and a potential C-terminal trans-membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N-terminal signal peptide sequences and C-terminal trans-membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae.

Caspases in developmental cell death.

Caspases are a family of evolutionarily conserved cysteine proteases that constitute the effector arm of the apoptotic machinery. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse point to evolutionarily conserved caspase function in developmentally programmed cell death in metazoans. Whereas in the nematode all developmental cell death is mediated by a single caspase, in Drosophila and the mouse some caspases appear to regulate cell death in a spatio-temporally restricted manner. This article reviews what we currently know about the roles of various caspases in the execution of developmentally programmed cell death and what may be expected from future research in this field.

Conversion of procaspase-3 to an autoactivating caspase by fusion to the caspase-2 prodomain.

Caspases are cysteine proteases that play an essential role in apoptosis. Initial activation of caspases defines the key step in apoptotic execution. Based on primary structure, caspases can be divided into two groups, those with long amino-terminal prodomains (class I), and those with relatively short prodomains (class II). On overexpression in mammalian cells, class I caspases can induce cell death that is dependent on their autocatalytic activity. Recent studies suggest that the long prodomains in some class I caspases are able to mediate dimerization of procaspase molecules, thereby promoting autoprocessing. In this communication, we demonstrate that fusion of the prodomain of a class I caspase (Nedd2/caspase-2) with procaspase-3 greatly augments autocatalysis and apoptosis induction by the chimeric caspase-3 molecule. The chimeric caspase-3 molecules were able to form homodimers in Saccharomyces cerevisiae and were efficiently processed in transfected mammalian cells. These results provide direct evidence for a role of a class I caspase prodomain in caspase autoactivation and processing and establish a basis for functional hierarchy among the two classes of caspases.

Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy.

Embryonic stem cells are pluripotent cells derived from the early mouse embryo that can be propagated stably in the undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in an embryonic and adult mouse in vivo, and can be induced to differentiate into many cell types in vitro. Exploitation of ES cell technology for the creation of mice bearing predetermined genetic alterations has received widespread attention because of the sophistication that it brings to the study of gene function in mammals. Analysis of cell differentiation in vitro has also been of value, leading to the identification of novel bioactive factors and the elucidation of cell specification mechanisms. In this paper, we summarise the features of pluripotent cell lines and their applications, foreshadowing the impact that these systems may have on human biology. While the isolation of definitive human pluripotent cell lines has not yet been achieved, potential applications for these cells in the study of human biology, particularly cell specification, can be envisaged. Of particular interest is the possibility that human embryonic stem cells with properties similar to mouse embryonic stem cells might provide a generic system for gene therapy.

Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells.

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. We describe a modified approach, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kb of homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for abeta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. Our results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes.

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.