Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells.

BACKGROUND: New therapies are required for castrate-resistant prostate cancer (CRPC), and growth-arrest specific 5 (GAS5) lncRNA, which riborepresses androgen receptor action, may offer novel opportunities in this regard. This lncRNA promotes the apoptosis of prostate cancer cells and its levels decline as prostate cancer cells acquire castrate-resistance, so that enhancing GAS5 expression may improve the effectiveness of chemotherapies. Since GAS5 is a member of the 5' terminal oligopyrimidine gene family, we have examined mTOR inhibition as a strategy to increase GAS5 expression. Furthermore, we have determined if GAS5 itself mediates the action of mTOR inhibitors, as demonstrated for other chemotherapeutic agents in prostate cancer cells. METHODS: The effects of mTOR inhibitors on GAS5 lncRNA levels and cell growth were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNAs and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. RESULTS: First generation mTORC1, combined mTORC1/mTORC2 and dual PI3K/mTOR inhibitors all increased cellular GAS5 levels and inhibited culture growth in androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU 145) cell lines. The latter exhibited low endogenous GAS5 expression, and GAS5 silencing in LNCaP and 22Rv1 cells decreased the sensitivity to mTOR inhibitors, whereas transfection of GAS5 lncRNA sensitized PC-3 and DU 145 cells to these agents. CONCLUSION: mTOR inhibition enhances GAS5 transcript levels in certain prostate cancer cell lines. This selectivity is likely to be related to endogenous GAS5 expression levels, since GAS5 lncRNA is itself required for mTOR inhibitor action in prostate cancer cells.

Regulation of apoptosis by long non-coding RNA GAS5 in breast cancer cells: implications for chemotherapy.

The putative tumour suppressor and apoptosis-promoting gene, growth arrest-specific 5 (GAS5), encodes long ncRNA (lncRNA) and snoRNAs. Its expression is down-regulated in breast cancer, which adversely impacts patient prognosis. In this preclinical study, the consequences of decreased GAS5 expression for breast cancer cell survival following treatment with chemotherapeutic agents are addressed. In addition, functional responses of triple-negative breast cancer cells to GAS5 lncRNA are examined, and mTOR inhibition as a strategy to enhance cellular GAS5 levels is investigated. Breast cancer cell lines were transfected with either siRNA to GAS5 or with a plasmid encoding GAS5 lncRNA and the effects on breast cancer cell survival were determined. Cellular responses to mTOR inhibitors were evaluated by assaying culture growth and GAS5 transcript levels. GAS5 silencing attenuated cell responses to apoptotic stimuli, including classical chemotherapeutic agents; the extent of cell death was directly proportional to cellular GAS5 levels. Imatinib action in contrast, was independent of GAS5. GAS5 lncRNA promoted the apoptosis of triple-negative and oestrogen receptor-positive cells but only dual PI3K/mTOR inhibition was able to enhance GAS5 levels in all cell types. Reduced GAS5 expression attenuates apoptosis induction by classical chemotherapeutic agents in breast cancer cells, providing an explanation for the relationship between GAS5 expression and breast cancer patient prognosis. Clinically, this relationship may be circumvented by the use of GAS5-independent drugs such as imatinib, or by restoration of GAS5 expression. The latter may be achieved by the use of a dual PI3K/mTOR inhibitor, to improve apoptotic responses to conventional chemotherapies.

Candidate tumour suppressor Fau regulates apoptosis in human cells: an essential role for Bcl-G.

FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.

A critical role for non-coding RNA GAS5 in growth arrest and rapamycin inhibition in human T-lymphocytes.

Non-coding RNA GAS5 (growth arrest-specific transcript 5) is a 5'-TOP (5'-terminal oligopyrimidine tract) RNA, whose translation, and consequently also stability, is controlled by the mTOR (mammalian target of rapamycin) pathway. GAS5 was identified by functional expression cloning and is necessary and sufficient for normal growth arrest in both leukaemic and untransformed human T-lymphocytes. GAS5 is also required for the inhibitory effects of rapamycin and its analogues on T-cells. The striking functional effects of GAS5 may be mediated through the snoRNAs (small nucleolar RNAs) encoded in its introns and/or through the unusual folding of the mRNA itself, which sequesters, and therefore inhibits, the glucocorticoid receptor.

FAU regulates carboplatin resistance in ovarian cancer.

The development of chemotherapy resistance by cancer cells is complex, using different mechanisms and pathways. The gene FAU (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified through functional expression cloning and previous data have shown that overexpression enhances apoptosis in several cell types. We demonstrate that the expression of FAU was reduced in the A2780cis (cisplatin resistant subclone of A2780) cell line compared with the A2780 ovarian cancer cell line, and was directly related to the cell line's sensitivity to carboplatin. Downregulation of FAU in the A2780 cell line by transfection with two predesigned short-interfering RNAs (siRNAs) to FAU resulted in a significant increase in resistance to carboplatin-induced cell death. Downregulation resulted in increased cell viability and reduced apoptosis after 72 hr of drug treatment compared with the negative controls (Kruskal-Wallis P = 0.0002). Transfection of the A2780cis cell line with the pcDNA3 plasmid containing FAU was associated with increased sensitivity to carboplatin-induced apoptosis, with decreased cell viability and increased apoptosis (Mann Whitney P < 0.0001). The expression of FAU was examined by quantitative real-time reverse transcriptase polymerase chain reaction in normal and malignant ovarian tissue. A significant reduction in the expression of FAU was seen in the malignant compared with normal ovarian samples (Kruskal-Wallis P = 0.0261). These data support a role for FAU in the regulation of platinum-resistance in ovarian cancer. Further research is needed into the apoptotic pathway containing FAU to investigate the potential for targeted therapies to increase or restore the platinum sensitivity of ovarian cancer.

Inhibition of human T-cell proliferation by mammalian target of rapamycin (mTOR) antagonists requires noncoding RNA growth-arrest-specific transcript 5 (GAS5).

The central importance of the serine/threonine protein kinase mTOR (mammalian Target of Rapamycin) in the control of cell growth and proliferation is well established. However, our knowledge both of the upstream pathways controlling mTOR activity and of the downstream events mediating these effects is still seriously incomplete. We report a previously unsuspected role for the nonprotein-coding RNA GAS5 in the inhibition of T-cell proliferation produced by mTOR antagonists such as rapamycin. GAS5 transcripts are up-regulated during growth arrest and after rapamycin treatment, and GAS5 has recently been shown to be necessary and sufficient for normal T-cell growth arrest. Down-regulation of GAS5 using RNA interference protects both leukemic and primary human T cells from the inhibition of proliferation produced by mTOR antagonists. The GAS5 transcript is a member of the 5' terminal oligopyrimidine class of RNAs, which is specifically controlled at the level of translation by the mTOR pathway, and the effects of GAS5 on the cell cycle provide a novel and important link to the control of proliferation. These observations point to a significant advance in our understanding of the mechanism of action of mTOR inhibitors, which is likely to lead to improvements in immunosuppressive and cancer therapy.

Apoptosis regulators Fau and Bcl-G are down-regulated in prostate cancer.

BACKGROUND: The molecular control of cell death through apoptosis is compromised in prostate cancer cells, resulting in inappropriate cell survival and resistance to cytotoxic therapy. Reduced expression of the functionally connected apoptosis-regulators and candidate tumor suppressors Fau and Bcl-G has recently been implicated in oncogenesis in other tissues. The present study examines the hypothesis that reduced expression of these genes may be involved in prostate cancer. METHODS: Fau and Bcl-G mRNA levels were determined by real time RT-PCR in two independent prostate tissue collections. In experiments in vitro, Fau and Bcl-G levels in prostate cancer cell lines were reduced using RNA interference and the effects on sensitivity to UVC irradiation were determined. RESULTS: Fau and Bcl-G mRNA levels were both lower in prostate cancer tissue than in normal prostate and Benign Prostate Hyperplasia. Active down-regulation of Fau and Bcl-G expression in vitro resulted in decreased sensitivity to UVC-induced cytotoxicity. Simultaneous down-regulation of Fau and Bcl-G produced a decrease in sensitivity which was similar to either gene alone. CONCLUSIONS: Fau and Bcl-G mRNA levels are both decreased in prostate cancer. In prostate cancer cell lines in vitro such down-regulation results in reduced sensitivity to UVC-induced cytotoxicity, consistent with the putative roles of these genes as candidate prostate tumor suppressors. The absence of an additive effect when Fau and Bcl-G were down-regulated simultaneously is consistent with the two genes acting in the same apoptosis pathway, for example, with the pro-apoptotic effects of Fau being mediated through modulation of Bcl-G.

Protein phosphatase 4 regulates apoptosis, proliferation and mutation rate of human cells.

The proteins which regulate apoptosis are of great importance both in normal cell biological processes and in the development of pathology in the diverse diseases which involve apoptosis dysfunction. The activity of many of these proteins is controlled by reversible phosphorylation, so that the relevant kinases and phosphatases play crucial roles in apoptosis control. Here we report the analysis of the role of the serine/threonine protein phosphatase, protein phosphatase 4, in controlling the apoptosis of HEK 293 T cells, using the complementary techniques of gene over-expression and down regulation through RNA interference. This analysis has demonstrated that PP4 regulates both apoptosis and proliferation in human cells and has also shown that the level of PP4 has a strong influence on gene mutation rate, which is crucial to oncogenesis. A parallel proteomic analysis has shown that the phosphorylation status of many relevant protein targets is affected by changes in PP4 and has focused attention particularly on the critical apoptosis regulators Bad and PEA-15. The phosphorylation of both of these proteins is increased when PP4 levels are suppressed, and is reduced when PP4 levels are increased, with striking consequences for the fate of the cell.

Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer.

INTRODUCTION: Programmed cell death through apoptosis plays an essential role in the hormone-regulated physiological turnover of mammary tissue. Failure of this active gene-dependent process is central both to the development of breast cancer and to the appearance of the therapy-resistant cancer cells that produce clinical relapse. Functional expression cloning in two independent laboratories has identified Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene (Fau) as a novel apoptosis regulator and candidate tumour suppressor. Fau modifies apoptosis-controller Bcl-G, which is also a key target for candidate oncoprotein maternal embryonic leucine zipper kinase (MELK). METHODS: We have used RNA interference to downregulate Fau and Bcl-G expression, both simultaneously and independently, in breast cancer cells in vitro to determine the importance of their roles in apoptosis. Expression of Fau, Bcl-G and MELK was measured by quantitative RT-PCR in breast cancer tissue and in matched breast epithelial tissue from the same patients. Expression data of these genes obtained using microarrays from a separate group of patients were related to patient survival in Kaplan-Meier analyses. RESULTS: siRNA-mediated downregulation of either Fau or Bcl-G expression inhibited apoptosis, and the inhibition produced by combining the two siRNAs was consistent with control of Bcl-G by Fau. Fau expression is significantly reduced in breast cancer tissue and this reduction is associated with poor patient survival, as predicted for a candidate breast cancer tumour suppressor. In addition, MELK expression is increased in breast cancer tissue and this increase is also associated with poor patient survival, as predicted for a candidate oncogene. Bcl-G expression is reduced in breast cancer tissue but decreased Bcl-G expression showed no correlation with survival, indicating that the most important factors controlling Bcl-G activity are post-translational modification (by Fau and MELK) rather than the rate of transcription of Bcl-G itself. CONCLUSIONS: The combination of in vitro functional studies with the analysis of gene expression in clinical breast cancer samples indicates that three functionally interconnected genes, Fau, Bcl-G and MELK, are crucially important in breast cancer and identifies them as attractive targets for improvements in breast cancer risk prediction, prognosis and therapy.

Retroviral insertional mutagenesis implicates E3 ubiquitin ligase RNF168 in the control of cell proliferation and survival.

The E3 ubiquitin ligase RNF168 is a ring finger protein that has previously been identified to play an important regulatory role in the repair of double-strand DNA breaks.  In the present study, an unbiased forward genetics functional screen in mouse granulocyte/ macrophage progenitor cell line FDCP1 has identified E3 ubiquitin ligase RNF168 as a key regulator of cell survival and proliferation. Our data indicate that RNF168 is an important component of the mechanisms controlling cell fate, not only in human and mouse haematopoietic growth factor-dependent cells, but also in the human breast epithelial cell line MCF-7. These observations therefore suggest that RNF168 provides a connection to key pathways controlling cell fate, potentially through interaction with PML nuclear bodies and/or epigenetic control of gene expression. Our study is the first to demonstrate a critical role for RNF168 in the in the mechanisms regulating cell proliferation and survival, in addition to its well-established role in DNA repair.

Functional expression cloning reveals a central role for the receptor for activated protein kinase C 1 (RACK1) in T cell apoptosis.

Mammalian cDNA expression cloning was used to identify novel genes that regulate apoptosis. Using a functional screen, we identified a partial cDNA for the receptor for activated protein kinase C 1 (RACK1) through selection for resistance to phytohemagglutinin and gamma-irradiation. Expression of this partial cDNA in T cell lines using a mammalian expression vector produced an increase in RACK1 expression and resulted in resistance to dexamethasone- and ultraviolet-induced apoptosis. Down-regulation of RACK1 using RNA interference abolished the resistance of the transfected cells to apoptosis. Overexpression of full-length RACK1 also resulted in the suppression of apoptosis mediated by several apoptotic stimuli, and this effect was quantitatively consistent with the effects of the original cDNA isolated on endogenous RACK1 levels. Together, these findings suggest that RACK1 plays an important role in the intracellular signaling pathways that lead to apoptosis in T cells.

The antiapoptotic RBM5/LUCA-15/H37 gene and its role in apoptosis and human cancer: research update.

The candidate tumour-suppressor gene, LUCA-15/RBM5/H37, maps to the lung cancer tumour-suppressor locus 3p21.3. The LUCA-15 gene locus encodes at least four alternatively spliced transcripts that have been shown to function as regulators of apoptosis, a fact which may have major significance in tumour regulation. This review highlights recent evidence that further implicates the LUCA-15 locus in the control of apoptosis and cell proliferation, and focuses on the observations that confirm the tumour-suppressor activity of this gene.

The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells.

Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and -insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies.

Regulation of apoptosis by fau revealed by functional expression cloning and antisense expression.

Functional expression cloning is a powerful strategy for identifying critical steps in biological pathways independently of prior assumptions. It is particularly suitable for the identification of molecules crucial to the control of apoptosis. Our screen for sequences suppressing T-cell apoptosis isolated a sequence antisense to fau (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene). The fox gene in FBR murine osteosarcoma virus is also antisense to fau and several reports have indicated that fau displays tumour suppressor and oncogenic properties in different contexts. Our observations indicate that the fau antisense sequence suppresses expression of endogenous fau mRNA and produces resistance to apoptosis induced both by the glucocorticoid analogue dexamethasone' by ultraviolet radiation, and by the anticancer drug cisplatin. In all cases, colony-forming ability is protected, indicating that fau affects the critical events prior to commitment to cell death. Overexpression of fau in the sense orientation induces cell death, which is inhibited both by Bcl-2 and by inhibition of caspases, in line with its proposed role in apoptosis.

Complete cDNA sequence of SAP-like pentraxin from Limulus polyphemus: implications for pentraxin evolution.

The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology.

Isolation and characterization of a novel pituitary tumor apoptosis gene.

To determine mechanisms for pituitary neoplasia we used methylation-sensitive arbitrarily primed-PCR to isolate novel genes that are differentially methylated relative to normal pituitary. We report the isolation of a novel differentially methylated chromosome 22 CpG island-associated gene (C22orf3). Sodium bisulfite sequencing of pooled tumor cohorts, used in the isolation of this gene, showed that only a proportion of the adenomas within the pools were methylated; however, expression analysis by quantitative RT-PCR of individual adenoma irrespective of subtype showed the majority (30 of 38; 79%) failed to express this gene relative to normal pituitary. Sodium bisulfite sequencing of individual adenomas showed that 6 of 30 (20%) that failed to express pituitary tumor apoptosis gene (PTAG) were methylated; however, genetic change as determined by loss of heterozygosity and sequence analysis was not apparent in the remaining tumors that failed to express this gene. In those cases where the CpG island of these genes was methylated it was invariably associated with loss of transcript expression. Enforced expression of C22orf3 in AtT20 cells had no measurable effects on cell proliferation or viability; however, in response to bromocriptine challenge (10-40 microm) cells expressing this gene showed a significantly augmented apoptotic response as determined by both acridine orange staining and TUNEL labeling. The apoptotic response to bromocriptine challenge was inhibited in coincubation experiments with the general caspase inhibitor z-VAD-fmk. In addition, in time course experiments, direct measurement of active caspases by fluorochrome-labeled inhibition of caspases, showed an augmented increase (approximately 2.4 fold) in active caspases in response to bromocriptine challenge in cells expressing C22orf3 relative to those harboring an empty vector control. The pituitary tumor derivation and its role in apoptosis of this gene led us to assign the acronym PTAG to this gene and its protein product. The ability of cells, showing reduced expression of PTAG, to evade or show a blunted apoptotic response may underlie oncogenic transformation in both the pituitary and other tumor types.

Molecular and Cellular Mechanisms of Action of Tumour Suppressor GAS5 LncRNA.

It is increasingly recognised that lncRNAs play essential regulatory roles in fundamental biological processes and, consequently, that their dysregulation may contribute to major human diseases, including cancer. Better understanding of lncRNA biology may therefore offer new insights into pathogenetic mechanisms and thereby offer novel opportunities for diagnosis and therapy. Of particular interest in this regard is GAS5 lncRNA, which is down-regulated in multiple cancers, with expression levels related to both clinico-pathological characteristics and patient prognosis. Functional studies have further shown that GAS5 lncRNA both inhibits the proliferation and promotes the apoptosis of multiple cell types, and that together these cellular mechanisms of action are likely to form the basis of its tumour suppressor action. At the same time, advances have been made in our understanding of the molecular mechanisms of GAS5 lncRNA action in recent years, including riborepression of certain steroid hormone receptors and sequestration of miR-21, impacting key regulatory pathways of cell survival. Overall this accumulating knowledge has the potential to improve both the diagnosis and treatment of cancer, and ultimately patient outcome.

Apoptosis in the malaria protozoan, Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito.

Death by apoptosis regulates cell numbers in metazoan tissues and it is mediated by activation of caspases and results in characteristic morphological and biochemical changes. We report here that the malaria protozoan, Plasmodium berghei, exhibits features typical of metazoan apoptotic cells including condensation of chromatin, fragmentation of the nuclear DNA and movement of phosphatidylserine from the inner to the outer lamellae of the cell membrane. In addition, proteins with caspase-like activity were identified in the cytoplasm of the ookinete suggesting that the cellular mechanism of cell death may be similar to that of multicellular eukaryotes. Our data show that more than 50% of the mosquito midgut stages of the parasite die naturally by apoptosis before gut invasion. Cell death was prevented by a caspase inhibitor, treatment resulting in a doubling of parasite intensity. All these features also occur in vitro. Cell suicide thus plays a major and hitherto unrecognised role in controlling parasite populations and could be a novel target for malaria control strategies.

RBM5/LUCA-15--tumour suppression by control of apoptosis and the cell cycle?

The candidate tumour suppressor gene, LUCA-15, maps to the lung cancer tumour suppressor locus 3p21.3. The LUCA-15 gene locus encodes at least four alternatively spliced transcripts, which have been shown to function as regulators of apoptosis, a fact that may have a major significance in tumour regulation. This review highlights evidence that implicates the LUCA-15 locus in the control of apoptosis and cell proliferation, and reports observations that significantly strengthen the case for tumour suppressor activity by this gene.

Apoptosis gene hunting using retroviral expression cloning: identification of vacuolar ATPase subunit E.

Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.