Mammalian SIRT1 is an NAD-dependent deacetylase with critical roles in the maintenance of homeostasis and cell survival. Elevated levels of SIRT1 protein are evident in cancer in which SIRT1 can function as a cancer-specific survival factor. Here we demonstrate that elevated SIRT1 protein in human cells is not attributable to increased SIRT1 mRNA levels but, instead, reflects SIRT1 protein stability. RNAi-mediated depletion of JNK2 reduced the half-life of SIRT1 protein from >9 h to <2 h and this correlated with lack of SIRT1 protein phosphorylation at serine 27. In contrast, depletion of JNK1 had no effect upon SIRT1 protein stability and SIRT1 phosphorylation at serine 47 showed no correlation with SIRT1 protein stability. Thus we show that JNK2 is linked, directly or indirectly, with SIRT1 protein stability and that this function is coupled with SIRT1 phosphorylation at serine 27. Our observations identify a route for therapeutic modulation of SIRT1 protein levels in SIRT1-linked diseases including cancer, neurodegeneration and diabetes.

SIRT3, one of seven mammalian sirtuins, is a NAD-dependent deacetylase. SIRT3 localizes to mitochondria where it deacetylates and thus activates acetyl-CoA synthetase 2 (AceCS2), indicating a role for SIRT3 in metabolism. Here we provide evidence that SIRT3 also impacts upon apoptosis and cell growth control. Using RNAi under basal (non-stress) conditions we show that SIRT3 is required for apoptosis induced by selective silencing of Bcl-2 in HCT116 human epithelial cancer cells. Identical treatment of ARPE19 epithelial non-cancer cells induces G(1) growth arrest which also proved to be SIRT3-dependent. Previously we have identified SIRT1 and JNK2 as constitutive suppressors of apoptosis in HCT116 cells. We now demonstrate that SIRT3 functions in JNK2-regulated apoptosis but is dispensable for SIRT1-regulated apoptosis. SIRT3 is also dispensable for stress-induced apoptosis. Thus the pro-apoptotic functioning of SIRT3 is selectively coupled with defined pathways regulating cell survival under basal conditions.

Most transformed cells display abnormally high levels of RNA polymerase (pol) III transcripts. Although the full significance of this is unclear, it may be fundamental because healthy cells use two key tumor suppressors to restrain pol III activity. We present the first evidence that a pol III transcription factor is overexpressed in tumors. This factor, TFIIIC2, is a histone acetyltransferase that is required for synthesis of most pol III products, including tRNA and 5S rRNA. TFIIIC2 is a complex of five polypeptides, and mRNAs encoding each of these subunits are overexpressed in human ovarian carcinomas; this may explain the elevated TFIIIC2 activity that is found consistently in the tumors. Deregulation in these cancers is unlikely to be a secondary response to rapid proliferation, because there is little or no change in TFIIIC2 mRNA levels when actively cycling cells are compared with growth-arrested cells in culture. Using purified factors, we show that raising the level of TFIIIC2 is sufficient to stimulate pol III transcription in ovarian cell extracts. The data suggest that overexpression of TFIIIC2 contributes to the abnormal abundance of pol III transcripts in ovarian tumors.ovarian cancer ͉ pol III R NA polymerase (pol) III synthesizes several essential products, including tRNA, 5S rRNA, 7SL RNA, and U6 snRNA (1). It is well established that pol III products are overexpressed in many cell lines transformed by DNA tumor viruses, RNA tumor viruses, or chemical carcinogens (e.g., refs. 2-6). These observations also apply to tumors in vivo (7,8). Thus, Northern blots showed that 7SL RNA is abnormally abundant in every tumor analyzed, relative to normal tissue from the same patient (8). Furthermore, in situ hybridization of breast, lung, and tongue carcinomas revealed increased levels of pol III transcripts in neoplastic cells relative to the surrounding healthy tissue (7,8).To maintain a constant size, a cell must duplicate its components before division. Because most of a cell's dry mass is protein, a high rate of protein synthesis is a prerequisite of rapid growth. Indeed, growth rate is directly proportional to the rate of protein accumulation (9). A 50% reduction in protein synthesis causes cells to withdraw from cycle and quiesce (10, 11). The availability of tRNA and rRNA is clearly an important determinant of the rate of translation. High levels of pol III transcription are therefore necessary to sustain rapid growth. This may help explain the frequent deregulation of pol III in transformed cells. However, pol III is also activated in several slowly growing tumor cell types, such as the osteosarcoma line SAOS2 (12). This shows that the strong correlation between transformation and pol III activation is not simply a consequence of rapid growth.Although elevated pol III transcript levels are frequently observed in transformed cells, in most cases the mechanistic basis is unknown. A partial explanation was suggested by the discovery that the retinoblastoma protein RB can repress pol III (12-14). Overe...

The physical microenvironment of tumours is characterized by heterotypic cell interactions and physiological gradients of nutrients, waste products and oxygen. This tumour microenvironment has a major impact on the biology of cancer cells and their response to chemotherapeutic agents. Despite this, most in vitro cancer research still relies primarily on cells grown in 2D and in isolation in nutrient- and oxygen-rich conditions. Here, a microfluidic device is presented that is easy to use and enables modelling and study of the tumour microenvironment in real-time. The versatility of this microfluidic platform allows for different aspects of the microenvironment to be monitored and dissected. This is exemplified here by real-time profiling of oxygen and glucose concentrations inside the device as well as effects on cell proliferation and growth, ROS generation and apoptosis. Heterotypic cell interactions were also studied. The device provides a live ‘window’ into the microenvironment and could be used to study cancer cells for which it is difficult to generate tumour spheroids. Another major application of the device is the study of effects of the microenvironment on cellular drug responses. Some data is presented for this indicating the device’s potential to enable more physiological in vitro drug screening.

Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer's glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD+) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD+ and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD+. This effect was specific for p53+/+ cancer cells and correlated with (i) reduced activity of NAD+-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53+/+ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53+/+ cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD+ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.

has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.The retinoblastoma susceptibility gene encodes the important tumor suppressor retinoblastoma protein (RB) (12,15,37,57). Inactivating mutations in this gene are found in many human cancers, including retinoblastomas, many sarcomas, and bladder and small-cell lung carcinomas (12,15,37,57). In a large proportion of other human malignancies the Rb gene is of the wild type, but its function is disrupted. For example, the cyclin-dependent kinases that switch off RB are hyperactive in many tumors (12,15,37,57). Indeed, it has been suggested that the regulatory pathway involving RB may be compromised in all human malignancies (57). It is therefore of considerable importance to obtain a clear understanding of the mechanisms used by RB to influence cellular activity.RB is a highly abundant protein that has been shown to bind and regulate a variety of transcription factors (12,15,37,48). The best-characterized example is the factor E2F, which controls several genes that are transcribed by RNA polymerase (Pol) II (11,14). Indeed, RB was thought for some time to control only Pol II-transcribed genes. However, recent advances have demonstrated that RB can also regulate transcription by Pols I and III (7,58,62). Pol I synthesizes large rRNA, whereas Pol III synthesizes a variety of small stable RNAs, including 5S rRNA and tRNA; together Pols I and III can be responsible for up to 80% of all nuclear transcription (39).Experiments using knockout mice revealed a major role for endogenous RB in regulating Pol III. Primary fibroblasts from Rb Ϫ/Ϫ mice were found to have a fivefold higher Pol III transcriptional activity than equivalent cells from wild-type mice, when assayed in vitro or in vivo (28, 62). Furthermore, overexpression of RB can inhibit Pol III transcription in transfected cells or in a system reconstituted with partially purified factors (8,28,62). This repression involves binding of RB to the Pol III-specific factor TFIIIB (8, 28). TFIIIB is a multisubunit complex which contains the TATA-binding protein (TBP), a TBP-associated factor (TAF) called BRF, and at least one other esse...

BackgroundThe NAD-dependent deacetylase SIRT1 is a nutrient-sensitive coordinator of stress-tolerance, multiple homeostatic processes and healthspan, while p53 is a stress-responsive transcription factor and our paramount tumour suppressor. Thus, SIRT1-mediated inhibition of p53 has been identified as a key node in the common biology of cancer, metabolism, development and ageing. However, precisely how SIRT1 integrates such diverse processes remains to be elucidated.Methodology/Principal FindingsHere we report that SIRT1 is alternatively spliced in mammals, generating a novel SIRT1 isoform: SIRT1-ΔExon8. We show that SIRT1-ΔExon8 is expressed widely throughout normal human and mouse tissues, suggesting evolutionary conservation and critical function. Further studies demonstrate that the SIRT1-ΔExon8 isoform retains minimal deacetylase activity and exhibits distinct stress sensitivity, RNA/protein stability, and protein-protein interactions compared to classical SIRT1-Full-Length (SIRT1-FL). We also identify an auto-regulatory loop whereby SIRT1-ΔExon8 can regulate p53, while in reciprocal p53 can influence SIRT1 splice variation.Conclusions/SignificanceWe characterize the first alternative isoform of SIRT1 and demonstrate its evolutionary conservation in mammalian tissues. The results also reveal a new level of inter-dependency between p53 and SIRT1, two master regulators of multiple phenomena. Thus, previously-attributed SIRT1 functions may in fact be distributed between SIRT1 isoforms, with important implications for SIRT1 functional studies and the current search for SIRT1-activating therapeutics to combat age-related decline.

CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.Protein kinase CK2 (formerly known as casein kinase II) is ubiquitous and highly conserved in eukaryotes (reviewed in references 1 and 29). It phosphorylates proteins on serine and threonine in both the nucleus and the cytoplasm. CK2 exists as a tetramer, composed of two isozymic catalytic subunits, ␣ and ␣Ј, and two copies of a regulatory ␤ subunit or one copy each of ␤ and the closely related ␤Ј. The CK2␣ and CK2␣Ј subunits are nearly 90% identical and can compensate for each other, but there is also some functional specialization (57, 69). The ␤ subunits allow optimal kinase activity and can regulate substrate specificity; they form a stable dimer linking the two catalytic subunits, which do not contact each other (35).Although its signaling function has long remained obscure, CK2 has been shown recently to form part of the Wnt pathway in both Drosophila and mammals (54, 65). Many studies have found that increases in the level and/or activity of CK2 are associated with cell growth and proliferation (for example, references 3, 4, 7, 25, 30, 34, and 39). Thus, CK2 expression can be increased by mitogens (7, 39), and CK2 is most abundant in cells with high mitotic activity, such as transformed cells and normal colorectal mucosa (34). Indeed, microinjection of CK2 can induce immediate-early gene expression in the absence of growth factors (11). Conversely, inactivation of CK2 by specific antibodies or antisense oligonucleotides can arrest the proliferation of primary human fibroblasts (41,42). Similarly, cell cycle progression is blocked in Saccharomyces ...