LUCA-15/RBM5, a putative tumour suppressor, enhances multiple receptor-initiated death signals.

LUCA-15/RBM5 is a putative tumour suppressor. The gene encodes a number of alternative RNA splice variants with differing abilities to either enhance or suppress apoptosis, and it is likely that this ability to modulate apoptosis is central to the putative tumour suppressor activity of LUCA-15. This report demonstrates for the first time that expression from the LUCA-15 locus modulates apoptosis triggered by the death-inducing ligand TRAIL. Using Jurkat T lymphoblastic leukemia cells, LUCA-15 expression was shown to enhance not only TRAIL but TNF-alpha- and Fas-mediated apoptosis. LUCA-15, therefore, has the ability to lower the apoptotic threshold of multiple receptor-initiated death-inducing signals. Of note, sensitisation of the Jurkat cells to TRAIL was shown to depend on new protein synthesis, since no enhancement of apoptosis was observed when cells were exposed to both TRAIL and the protein synthesis inhibitor cycloheximide. This result suggests that LUCA-15 does not act independently to regulate apoptosis, but modulates a process that requires additional, newly synthesized protein. These results may explain the putative role of LUCA-15 as a tumour suppressor, suggesting that lack of functional LUCA-15 could provide the means by which malignant T cells escape receptor-initiated apoptotic signals.

Candidate tumour suppressor LUCA-15 can regulate multiple apoptotic pathways.

Functional screening of a human bone marrow cDNA library for suppressors of CD95-mediated apoptosis has led to the identification of a 326 bp fragment (Je2), which not only suppresses CD95-induced apoptosis in Jurkat T-cells, but maps to 3p21.3, to an intronic region of the candidate TSG LUCA-15 locus. Here we report that overexpression of Je2 in CEM-C7 T-cell line is able to suppress CD95-mediated apoptosis, and apoptosis induced by TNFalpha and the glucocorticoid analogue dexamethasone, but was not able to suppress death induced by the topoisomerase II inhibitor etoposide. Je2 inhibition of apoptosis is also associated with a change in the pattern of expression of LUCA-15-encoded proteins. Je2 might therefore function to inhibit apoptosis by destabilising message expression of LUCA-15 and promoting the degradation of its RNA and protein. This suppression of apoptosis by Je2 also appears to be associated with up-regulation of the apoptosis inhibitory protein Bcl-x(L). This study confirms that Je2 is a selective inhibitor of cell death and further implicates LUCA-15 gene locus in the control of apoptosis.

LUCA-15 suppresses CD95-mediated apoptosis in Jurkat T cells.

The candidate tumour suppressor gene, LUCA-15, maps to the lung cancer tumour suppressor locus 3p21.3. Overexpression of an alternative RNA splice variant of LUCA-15 has been shown to retard human Jurkat T cell proliferation and to accelerate CD95-mediated apoptosis. An antisense cDNA to the 3'-UTR of this splice variant was able to suppress CD95-mediated apoptosis. Here, we report that overexpression of LUCA-15 itself suppresses CD95-mediated apoptosis in Jurkat cells. This suppression occurs prior to the final execution stage of the CD95 signalling pathway, and is associated with up-regulation of the apoptosis inhibitory protein Bcl-2. LUCA-15 overexpression is also able to inhibit apoptosis induced by the protein kinase inhibitor staurosporine, but is not able to significantly suppress apoptosis mediated by the topoisomerase II inhibitor etoposide. These findings suggest that LUCA-15 is a selective inhibitor of cell death, and confirm the importance of the LUCA-15 genetic locus in the control of apoptosis.

LUCA-15-encoded sequence variants regulate CD95-mediated apoptosis.

Using an expression cloning system to discover novel genes involved in apoptosis, we identified a 326 bp bone marrow cDNA fragment (termed Je2) that suppresses, upon transfection, CD95-mediated apoptosis in Jurkat T cells. Sequence homology revealed that Je2 maps to 3p21.3, to an intronic region of the candidate TSG LUCA-15 locus. It represents, in fact, an antisense transcript to the 3'-UTR of two novel splice variants of this gene. Overexpression of sequence representing one of these splice variants (a 2.6 kb cDNA termed Clone 26), inhibited proliferation of Jurkat cells and sensitized them to CD95-mediated apoptosis. This study therefore implicates the LUCA-15 gene locus in the control of apoptosis.

Zinc has no effect on IL-3-mediated apoptosis of BAF-3 cells but enhances CD95-mediated apoptosis of jurkat cells.

The feasibility of using a zinc-inducible gene expression system for the study of apoptosis-controlling genes in BAF-3 murine B cells and Jurkat human T cells was evaluated. Initially, cell sensitivity to a range of zinc concentrations was examined. It was found that zinc concentrations above 60 microM were toxic to BAF-3 cells and those above 50 microM were toxic to Jurkat cells. Secondly, the zinc concentration required to achieve maximal gene expression was examined. BAF-3 cells transiently transfected with the pMTCB6+/luciferase vector were exposed to zinc concentrations ranging from 0-120 microM, whilst stably transfected Jurkat cells were exposed to 0-70 microM zinc. At zinc concentrations nontoxic to each cell type, the maximum induction achieved was 20-fold (at 60 microM) in BAF-3 cells, and 7.5-fold (at 50 microM) in Jurkat cells. Thirdly, the effect of zinc on apoptosis was examined. It was shown that exposure to nontoxic zinc concentrations had no effect on IL-3 withdrawal-mediated apoptosis of BAF-3 cells. However, in the case of Jurkat cells, pre-exposure to zinc augmented CD95-mediated apoptosis. These results illustrate the importance of characterizing individual cell lines when using zinc-inducible gene expression systems.

Viral promoter expression in CEM-C7 and Jurkat human T-lymphoid cell lines.

The strength of various viral promoters was examined in human T lymphoblastoid cell lines. CMV, RSV and SV40 promoter based-luciferase constructs were transiently transfected into CEM-C7 and Jurkat cells in order to compare promoter strength in each cell type. It was found that the CMV promoter was 10-fold stronger than the RSV promoter in Jurkat cells, but equivalent to the RSV promoter in CEM-C7 cells. Both the CMV and RSV promoters were significantly stronger than the SV40 promoter in Jurkat and CEM-C7 cells. In summary, promoter strengths are cell line specific (Jurkat: CMV > RSV >> SV40; CEM-C7: RSV = CMV >> SV40) and care must be exercised in choosing promoters intended to provide overexpression of chimeric genes.

Apoptosis: molecular regulation of cell death.

The field of apoptosis is unusual in several respects. Firstly, its general importance has been widely recognised only in the past few years and its surprising significance is still being evaluated in a number of areas of biology. Secondly, although apoptosis is now accepted as a critical element in the repertoire of potential cellular responses, the picture of the intra-cellular processes involved is probably still incomplete, not just in its details, but also in the basic outline of the process as a whole. It is therefore a very interesting and active area at present and is likely to progress rapidly in the next two or three years. This review emphasises recent work on the molecular mechanisms of apoptosis and, in particular, on the intracellular interactions which control this process. This latter area is of crucial importance since dysfunction of the normal control machinery is likely to have serious pathological consequences, probably including oncogenesis, autoimmunity and degenerative disease. The genetic analysis of programmed cell death during the development of the nematode Caenorhabditis elegans has proved very useful in identifying important events in the cell death programme. Recently defined genetic connections between C. elegans cell death and mammalian apoptosis have emphasised the value of this system as a model for cell death in mammalian cells, which, inevitably, is more complex. The signals inducing apoptosis are very varied and the same signals can induce differentiation and proliferation in other situations. However, some pathways appear to be of particular significance in the control of cell death; recent analysis of the apoptosis induced through the cell-surface Fas receptor has been especially important for immunology. Two gene families are dealt with in particular detail because of their likely importance in apoptosis control. These are, first, the genes encoding the interleukin-1 beta-converting enzyme family of cysteine proteases and, second, those related to the proto-oncogene bcl-2. Both of these families are homologous to cell death genes in C. elegans. In mammalian cells the number of members of both families which have been identified is growing rapidly and considerable effort is being directed towards establishing the roles played by each member and the ways in which they interact to regulate apoptosis. Other genes with established roles in the regulation of proliferation and differentiation are also important in controlling apoptosis. Several of these are known proto-oncogenes, e.g. c-myc, or tumour suppressors, e.g. p53, an observation which is consistent with the importance of defective apoptosis in the development of cancer. Viral manipulation of the apoptosis of host cells frequently involves interactions with these cellular proteins. Finally, the biochemistry of the closely controlled cellular self-destruction which ensues when the apoptosis programme has been engaged is also very important. The biochemical changes involved in inducing phagocytosis of the apoptotic cell, for example, allow the process to be neatly integrated within the tissues, under physiological conditions. Molecular defects in this area too may have important pathological consequences.

An upstream activator sequence regulates the murine Pgk-1 promoter and binds multiple nuclear proteins.

The murine Pgk-1 gene is driven by a strong promoter that is regulated by a 304 bp upstream activator sequence (UAS). The activity of the UAS is high in undifferentiated embryonal carcinoma cells but declines when these cells are induced to differentiate with retinoic acid. The effect of the UAS on promoter activity is particularly striking when the activity of the Pgk-1 promoter is assayed following its integration into the genome, suggesting that it may function by regulating chromatin structure in the region of the core promoter. Three sites on the UAS bind nuclear proteins. Two of these sites bind factors present in both embryonal carcinoma cells and their differentiated derivatives whereas one site binds factors present only in differentiated cells. There appears to be both cooperation and antagonism in the binding of proteins to different sites in the UAS, suggesting that the activity of the Pgk-1 promoter is determined by the constellation of proteins assembled upstream of its transcription start site.

The mouse Pgk-1 gene promoter contains an upstream activator sequence.

The Pgk-1 gene encodes the housekeeping enzyme, 3-phosphoglycerate kinase, and is ubiquitously expressed. This gene resides on the X chromosome in mammals and is always expressed except where it is silenced along with most other genes on the inactive X chromosome of female somatic cells or male germ cells. The Pgk-1 promoter is in a region rich in nucleotides G and C. This promoter can efficiently drive high levels of expression of reporter genes such as E. coli lacZ and neo. We have determined that the 120 bp upstream of the transcription start site functions as a core promoter. Upstream of this is a 320 bp region which enhances transcription from the core promoter in an orientation and position independent fashion. This 320 bp region does not enhance transcription from the core promoter of the SV40 early region. Nuclear proteins bind to this 320 bp fragment although the restricted regions to which binding can be demonstrated with gel mobility shift assays suggests that the activity of the enhancer may be mediated by factors which bind at multiple sites each with low affinity.