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PubMed2005

RNA polyadenylation in Archaea: not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus.

The addition of poly(A) tails to RNA is a phenomenon common to all organisms examined so far. No homologues of the known polyadenylating enzymes are found in Archaea and little is known concerning the mechanisms of messenger RNA degradation in these organisms. Hyperthermophiles of the genus Sulfolobus contain a protein complex with high similarity to the exosome, which is known to degrade RNA in eukaryotes. Halophilic Archaea, however, do not encode homologues of these eukaryotic exosome components. In this work, we analysed RNA polyadenylation and degradation in the archaea Sulfolobus solfataricus and Haloferax volcanii. No RNA polyadenylation was detected in the halophilic archaeon H. volcanii. However, RNA polynucleotidylation occurred in hyperthermophiles of the genus Sulfolobus and was mediated by the archaea exosome complex. Together, our results identify the first organism without RNA polyadenylation and show a polyadenylation activity of the archaea exosome.

Exoribonucleases↗
PubMed2006

Subunit architecture of multimeric complexes isolated directly from cells.

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Chromatography, Affinity↗
PubMed2006

Structural and biochemical characterization of the yeast exosome component Rrp40.

The exosome is a protein complex that is important in both degradation and 3'-processing of eukaryotic RNAs. We present the crystal structure of the Rrp40 exosome subunit from Saccharomyces cerevisiae at a resolution of 2.2 A. The structure comprises an S1 domain and an unusual KH (K homology) domain. Close packing of the S1 and KH domains is stabilized by a GxNG sequence, which is uniquely conserved in exosome KH domains. Nuclear magnetic resonance data reveal the presence of a manganese-binding site at the interface of the two domains. Isothermal titration calorimetry shows that Rrp40 and archaeal Rrp4 alone have very low intrinsic affinity for RNA. The affinity of an archaeal core exosome for RNA is significantly increased in the presence of the S1-KH subunit Rrp4, indicating that multiple subunits might contribute to cooperative binding of RNA substrates by the exosome.

Amino Acid Motifs↗
PubMed1999

Tob2, a novel anti-proliferative Tob/BTG1 family member, associates with a component of the CCR4 transcriptional regulatory complex capable of binding cyclin-dependent kinases.

Human cDNAs encoding a novel member of Tob/BTG1 anti-proliferative family proteins were cloned. The putative protein product termed Tob2 consisted of 344 amino acids with high similarity to the Tob protein. The tob2 mRNA was 4.1 kb long and was ubiquitously expressed in human adult tissues, as was revealed by Northern blot hybridization. However, further in situ hybridization analysis showed a characteristic expression of the tob2 mRNA in oocytes, suggesting a unique role of Tob2 in oogenesis. Like the Tob protein, Tob2 inhibited cell cycle progression from the G0/G1 to S phases. Intriguingly, the amino-terminal half of Tob2 as well as that of Tob was associated with a human homologue of yeast Caf1, a component of the CCR4 transcription factor complex. Moreover, Caf1 was associated with cyclin dependent kinases. These data suggested that both Tob and Tob2 were involved in cell cycle regulation through their interaction with Caf1. Finally, the tob2 gene was mapped to human chromosome 22q13.1-q13.31.

3T3 Cells↗
PubMed2001

Regulation of the G2/M transition by p53.

p53 protects mammals from neoplasia by inducing apoptosis, DNA repair and cell cycle arrest in response to a variety of stresses. p53-dependent arrest of cells in the G1 phase of the cell cycle is an important component of the cellular response to stress. Here we review recent evidence that implicates p53 in controlling entry into mitosis when cells enter G2 with damaged DNA or when they are arrested in S phase due to depletion of the substrates required for DNA synthesis. Part of the mechanism by which p53 blocks cells at the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Cdc2 is inhibited simultaneously by three transcriptional targets of p53, Gadd45, p21, and 14-3-3 sigma. Binding of Cdc2 to Cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. p53 also represses the cdc2 gene, to help ensure that cells do not escape the initial block. Genotoxic stress also activates p53-independent pathways that inhibit Cdc2 activity, activation of the protein kinases Chk1 and Chk2 by the protein kinases Atm and Atr. Chk1 and Chk2 inhibit Cdc2 by inactivating Cdc25, the phosphatase that normally activates Cdc2. Chk1, Chk2, Atm and Atr also contribute to the activation of p53 in response to genotoxic stress and therefore play multiple roles. p53 induces transcription of the reprimo, B99, and mcg10 genes, all of which contribute to the arrest of cells in G2, but the mechanisms of cell cycle arrest by these genes is not known. Repression of the topoisomerase II gene by p53 helps to block entry into mitosis and strengthens the G2 arrest. In summary, multiple overlapping p53-dependent and p53-independent pathways regulate the G2/M transition in response to genotoxic stress.

14-3-3 Proteins↗
PubMed2001

p63alpha and DeltaNp63alpha can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes.

The p53 tumor suppressor protein plays a critical role in the regulation of the cell cycle and apoptosis. The importance of p53's functions is underscored by the high incidence of p53 mutations in human cancers. Recently, two p53-related proteins, p73 and p63, were identified as members of the p53 gene family. Multiple isoforms of p73 have been found, including DeltaN variants in which the N-termini are truncated. p63 is expressed as three major forms, p63alpha, p63beta and p63gamma, each of which differ in their C-termini. All three forms can be alternatively transcribed from a cryptic promoter located within intron 3, producing DeltaNp63alpha, DeltaNp63beta and DeltaNp63gamma. The high degree of similarity of p73 and p63 to evolutionarily conserved regions of p53 suggests that these proteins play an important and potentially redundant role in regulating cell cycle arrest and apoptosis. Here we describe the characterization of cell lines generated to inducibly express p63alpha and DeltaNp63alpha. We have found that p63alpha and DeltaNp63alpha can differentially regulate endogenous p53 target genes and induce cell cycle arrest and apoptosis. Deletion of the N-terminal 26 amino acids of DeltaNp63alpha abolished its ability to transactivate p53 target genes and induce cell cycle arrest and apoptosis. This indicates that a putative transactivation domain exists within the N-terminus of the DeltaN variants of p63. Furthermore, the differential regulation of p53 target genes by p63alpha and DeltaNp63alpha suggests that p63 and p53 utilize both similar and different signaling pathways to execute their cellular functions.

14-3-3 Proteins↗
PubMed2001

Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer.

We have identified 14-3-3 sigma (sigma) as a gene whose expression is lost in breast carcinomas, primarily by methylation-mediated silencing. In this report, we investigated the timing of loss of sigma gene expression during breast tumorigenesis in vivo. We analysed the methylation status of sigma in breast cancer precursor lesions using microdissection for selective tissue sampling. We found hypermethylation of sigma in 24 of 25 carcinomas (96%), 15 of 18 (83%) of ductal carcinoma in situ, and three of eight (38%) of atypical hyperplasias. None of the five hyperplasias without atypia showed sigma-hypermethylation. Unexpectedly, patients with breast cancer showed sigma hypermethylation in adjacent histologically normal breast epithelium, while this was never observed in individuals without evidence of breast cancer. Also, samples of periductal stromal breast tissue were consistently hypermethylated, underscoring the importance of selective tissue sampling for accurate assessment of 14-3-3-sigma methylation in breast epithelium. These results suggest that hypermethylation of 14-3-3-sigma occurs at an early stage in the progression to invasive breast cancer, and may occur in apparently normal epithelium adjacent to breast cancer. These results provide evidence that loss of expression of sigma is an early event in neoplastic transformation.

14-3-3 Proteins↗
PubMed2002

Coincident inactivation of 14-3-3sigma and p16INK4a is an early event in vulval squamous neoplasia.

The structure and expression of 14-3-3 sigma(sigma) was analysed in squamous carcinomas (SCC) of the vulva and in the vulval pre-malignant lesion vulval intraepithelial neoplasia (VIN). Sequence analysis of the sigma coding region did not detect mutations in any case of SCC or VIN III and loss of heterozygosity (LOH) occurred in only 2 out of 27 informative cases. In contrast to the absence of genetic change, methylation-specific PCR (MSP) analysis revealed dense CpG methylation within the sigma gene in approximately 60% of cases of vulval SCC, but methylation was not detected in matched, normal epithelial tissue. Methylation was associated in all cases with reduced or absent expression of sigma mRNA. There was no correlation between sigma methylation and HPV or p53 status. Analysis of pre-malignant vulval intraepithelial neoplasia (VIN) revealed that sigma methylation was detectable early in neoplastic development. Co-incident methylation, accompanied by loss of expression, of sigma and p16INK4a was commonly detected in both SCC and VIN III, suggesting that epigenetic silencing of these two genes is an early and important event in vulval neoplasia.

14-3-3 Proteins↗
PubMed2002

Frequent and histological type-specific inactivation of 14-3-3sigma in human lung cancers.

One isoform of the 14-3-3 family, 14-3-3sigma, plays a crucial role in the G2 checkpoint by sequestering Cdc2-cyclinB1 in the cytoplasm, and the expression of 14-3-3sigma is frequently lost in breast cancers. This loss of expression is thought to cause a G2 checkpoint defect, resulting in chromosomal aberrations. Since lung cancers frequently carry numerous chromosomal aberrations, we examined the DNA methylation status and expression level of the 14-3-3sigma gene in 37 lung cancer cell lines and 30 primary lung tumor specimens. We found that small cell lung cancer (SCLC) cell lines frequently showed DNA hypermethylation (9 of 13 lines, 69%), and subsequent silencing of the 14-3-3sigma gene. Among non-small cell lung cancers (NSCLC), large cell lung cancer cell lines showed frequent hypermethylation and silencing of 14-3-3sigma (4 or 7 lines, 57%). In contrast, in other NSCLC cell lines, hypermethylation occurred very rarely (1 of 17 lines, 6%). All eight primary SCLC specimens examined also showed a loss or significant reduction in 14-3-3sigma expression in vivo, while a loss or reduction of 14-3-3sigma expression was very rare in primary NSCLC specimens (1 of 22 tissues, 5%). This is the first description that indicates lung cancers frequently show significant inactivation of the 14-3-3sigma gene mainly due to DNA hypermethylation in SCLC, but rarely in NSCLC, suggesting involvement of the 14-3-3sigma gene in lung tumorigenesis in a histological type-specific manner.

14-3-3 Proteins↗
PubMed2002

Human p14(ARF)-mediated cell cycle arrest strictly depends on intact p53 signaling pathways.

The tumor suppressor ARF is transcribed from the INK4a/ARF locus in partly overlapping reading frames with the CDK inhibitor p16(Ink4a). ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19(ARF). Little is known about the dependency of human p14(ARF) function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14(ARF)-induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53(+/+)p21(CIP1+/+) 14-3-3sigma(+/+)), but not p53(-/-) counterparts, underwent G(1) and G(2) cell cycle arrest following infection with a p14(ARF)-adenovirus. In p21(CIP1-/-) cells, p14(ARF) did not induce G(1) or G(2) arrest, while 14-3-3sigma(-/-) counterparts were mainly arrested in G(1), pointing to essential roles of p21(CIP1) in G(1) and G(2) arrest and cooperative roles of p21 and 14-3-3sigma in ARF-mediated G(2) arrest. Our data demonstrate a strict p53 and p21(CIP1) dependency of p14(ARF)-induced cell cycle arrest in human cells.

14-3-3 Proteins↗
PubMed2002

Decreased expression of 14-3-3sigma in neuroendocrine tumors is independent of origin and malignant potential.

We recently reported that 14-3-3sigma is frequently inactivated in small cell lung cancer (SCLC) and a part of large cell carcinomas. Subsequent studies revealed that the large cell carcinomas could be morphologically categorized as large cell neuroendocrine carcinomas (LCNEC). The present study therefore examines 14-3-3sigma expression in a spectrum of neuroendocrine lung tumors, which had varied p53 status, proliferative activity and clinical aggressiveness. The expression of 14-3-3sigma was decreased in all four categories of the spectrum, (5 out of 5 typical carcinoids, 2 out of 2 atypical carcinoids, 5 out of 7 LCNECs and 15 out of 18 SCLCs). In sharp contrast, the level of 14-3-3sigma expression in 75 non-small cell lung cancers (NSCLCs) was the same as that in normal lung tissue, with only one exception. The expression status of neuroendocrine tumors and NSCLCs was not affected by p53 status, but dense promoter hypermethylation of the 14-3-3sigma gene was specifically observed in neuroendocrine tumors, suggesting that methylation plays a regulatory role in 14-3-3sigma expression in vivo as well as in vitro. Furthermore, the expression was not only down-regulated in pulmonary neuroendocrine tumors, but also in neuroendocrine tumors arising from various other organs, through examination of 123 non-pulmonary tumors. Since various carcinogenic machineries are involved in the neuroendocrine tumors, a reduced expression of 14-3-3sigma might be required for the development of neuroendocrine tumors. Constitutive 14-3-3sigma expression was distributed exclusively in putative stem cells of the normal lung, namely the basal cells of the bronchus, and type II pneumocytes. Notably, 14-3-3sigma expression was up-regulated during the regeneration of type II pneumocytes, suggesting that 14-3-3sigma plays a biological role when a regenerative and/or differentiating drive is activated, facilitating exit from stem cells.

14-3-3 Proteins↗
PubMed2003

Analysis of 14-3-3sigma expression in hyperproliferative skin diseases reveals selective loss associated with CpG-methylation in basal cell carcinoma.

The p53-regulated 14-3-3sigma gene encodes an inhibitor of cell cycle progression essential for senescence and clonal evolution of keratinocytes in vitro. Here we analysed the in vivo expression of 14-3-3sigma protein in several skin diseases, which are characterized by hyperproliferative keratinocytes. Unexpectedly, the 14-3-3sigma protein was expressed at high levels in psoriasis (11 of 11 patients), condylomata acuminata (11/11), actinic keratoses (11/11) and squamous cell carcinomas (SCC) (11/11). However, keratinocytes that had undergone transformation to basal cell carcinoma (BCC) showed partial (10 of 41; 24.4%) or complete (19 of 41; 46.3%) loss of 14-3-3sigma protein expression. BCC (5/5), SCC (6/6) and actinic keratoses (7/7) concomitantly expressed the p53-homolog p63 and 14-3-3sigma at high levels, ruling out potential inhibitory effects of p63 isoforms on 14-3-3sigma transcription as the basis for loss of 14-3-3sigma expression. Of 41 BCC samples isolated by laser-capture microdissection, 28 (68.3%) showed CpG-hypermethylation of the 14-3-3sigma promoter combined with reduced or absent 14-3-3sigma protein levels in 22 cases (78.6%). Since it has been reported that BCC retain wild-type p16(INK4A) and here BCC with CpG-methylation of 14-3-3sigma did not show CpG-methylation of p16(INK4A) (0/17), silencing of 14-3-3sigma may contribute to evasion of senescence in BCC. As experimental removal of 14-3-3sigma sensitizes to DNA damage, silencing of 14-3-3sigma may explain the high efficacy of radiation therapy in the treatment of BCC.

14-3-3 Proteins↗
PubMed2004

The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe.

Fusion between nonsynchronized cells leads to the formation of heterokarya which transiently activate Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 and enter the prophase of the cell cycle, where they arrest due to a loss of Cdk1/cyclin B1 activity, activate p53, disorganize centrosomes, and undergo apoptosis. Here, we show that the down regulation of Cdk1/cyclin B is secondary to the activation of the DNA structure checkpoint kinase Chk2. Thus, syncytia generated by the fusion of asynchronous HeLa cells contain elevated levels of active Chk2 but not Chk1. Chk2 bearing the activating phosphorylation on threonine-68 accumulates in BRCA1 nuclear bodies when the cells arrest at the G2/M boundary. Inhibition of Chk2 by transfection of a dominant-negative Chk2 mutant or a chemical inhibitor, debromohymenialdesine, stabilizes centrosomes, maintains high cyclin B1 levels, and allows for a prolonged activation of Cdk1. Under these conditions, multinuclear HeLa syncytia do not arrest at the G2/M boundary and rather enter mitotis and subsequently die during the metaphase of the cell cycle. This mitotic catastrophe is associated with the activation of the pro-apoptotic caspase-3. Inhibition of caspases allows the cells to go beyond the metaphase arrest, indicating that apoptosis is responsible for cell death by mitotic catastrophe. In another, completely different model of mitotic catastrophe, namely 14.3.3 sigma-deficient HCT116 colon carcinoma cells treated with doxorubicin, Chk2 activation was also found to be deficient as compared to 14.3.3 sigma-sufficient controls. Inhibition of Chk2 again facilitated the induction of mitotic catastrophe in HCT116 wild-type cells. In conclusion, a conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe, provided that the checkpoint kinase Chk2 is inhibited. Inhibition of Chk2 thus can sensitize proliferating cells to chemotherapy-induced apoptosis.

14-3-3 Proteins↗
PubMed2004

The exonuclease ISG20 is directly induced by synthetic dsRNA via NF-kappaB and IRF1 activation.

Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virus-host interactions. Recently, we demonstrated that the IFN-induced 3' --> 5' exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-kappaB factors p50 and p65 bind and activate the kappaB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-kappaB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.

Binding Sites↗
PubMed2004

Proteomic identification of 14-3-3 sigma as a common component of the androgen receptor and the epidermal growth factor receptor signaling pathways of the human prostate epithelial cell line M12.

The epidermal growth factor receptor and androgen receptor (AR) both play major roles in the control of prostate growth. Our hypothesis is that shared downstream components of these two signaling pathways are significant participants in androgen-independent growth. Our first objective was to identify proteins whose activation and/or expression in AR-positive prostate epithelial cells are induced by both epidermal growth factor (EGF) and dihydrotestosterone (DHT). AR expression was induced in a tumorigenic, metastatic subline of the SV40 large T-antigen immortalized human prostate epithelial subline M12 by stable transfection with human wild-type AR cDNA. These M12AR (+) cells with functional AR were treated in parallel with EGF (10 ng/ml) or DHT (10(-8) M) for 24 h before 2D gel electrophoresis and Western immunoblotting with antiphosphotyrosine monoclonal antibody. Coomassie blue-stained spots on a 2D gel run in parallel were aligned with the phosphoproteins on the Western immunoblot, and identified by matrix-assisted laser desorption ionization/time-of-flight mass spectroscopy. The most interesting of the seven proteins that appeared to be phosphorylated by these criteria was 14-3-3 protein sigma. Protein extracted after either EGF or DHT treatment, immunoprecipitated with antiphosphotyrosine monoclonal antibody, and immunoblotted by anti-14-3-3 sigma confirmed phosphorylation of 14-3-3 sigma. Addition of either DHT or EGF to the M12AR(+) cells induced subcellular migration of 14-3-3 sigma and activated a 14-3-3 sigma reporter construct. Immunohistochemical analysis revealed nuclear localization of 14-3-3 sigma in higher Gleason grade prostate cancers relative to benign glands. These findings implicate 14-3-3 sigma in the development of human prostate cancer cells and could provide a new target for intervention in prostate cancer.

14-3-3 Proteins↗
PubMed2004

Prostate cancer is characterized by epigenetic silencing of 14-3-3sigma expression.

In order to identify tumor suppressive genes silenced by CpG methylation in prostate carcinoma (PCa), we determined genome-wide expression changes after pharmacological reversal of CpG methylation-mediated transcriptional repression in three PCa cell lines using microarray analysis. Thereby, epigenetic silencing of the 14-3-3sigma gene was detected in the cell line LNCaP. 14-3-3sigma encodes a p53-regulated inhibitor of cell cycle progression. Laser microdissection was used to isolate different cell types present in diseased prostatic tissue. Subsequent methylation-specific PCR analysis showed CpG methylation of 14-3-3sigma in all 41 primary PCa samples analysed, which was accompanied by a decrease or loss of 14-3-3sigma protein expression. In contrast, normal prostate epithelial and benign prostate hyperplasia cells showed high levels of 14-3-3sigma expression. PCa-precursor lesions (prostatic intraepithelial neoplasia) also displayed decreased levels of 14-3-3sigma expression in luminal cells, which are known to contain shortened telomeres. RNA interference-mediated inactivation of 14-3-3sigma compromised a DNA damage-induced G(2)/M arrest in the PCa cell line PC3. The generality of CpG methylation and downregulation of 14-3-3sigma expression in PCa suggests that it significantly contributes to the formation of PCa, potentially by allowing the escape from a DNA damage-induced arrest elicited by telomere shortening.

14-3-3 Proteins↗
PubMed2006

CpG island promoter methylation and silencing of 14-3-3sigma gene expression in LNCaP and Tramp-C1 prostate cancer cell lines is associated with methyl-CpG-binding protein MBD2.

14-3-3sigma proteins regulate numerous cellular processes that are important to cancer development. One of its biological roles involves G2 cell-cycle arrest following DNA damage. It has also been reported that the loss of 14-3-3sigma expression via CpG methylation may contribute to malignant transformation by impairing the G2 cell-cycle checkpoint function, thereby allowing an accumulation of genetic defects. However, how the CpG methylation-dependent silencing mechanism works in relation to promoter methylation associated with methyl-CpG-binding proteins (MeCPs) is still unclear. To better understand the mechanism, we first examined the methylation status of the 14-3-3sigma promoter-associated CpG islands and 14-3-3sigma gene expression in a subset of prostate cancer cell lines using methylation-specific PCR (MSP), an HhaI-based DNA methylation assay, and reverse transcription-PCR (RT-PCR). We found that the 14-3-3sigma expression is lost in LNCaP and Tramp-C1 prostate cancer cell lines and that this expression is restored after treatment with epigenetic silencing modifiers 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). These results imply transcriptional silencing via promoter-associated CpG methylation. Chromatin immunoprecipitation analysis revealed that methyl-CpG-binding protein 2 (MBD2) is associated preferentially to the methylated CpG island in the 14-3-3sigma promoter in LNCaP and Tramp-C1 cells but not in 14-3-3sigma-expressing PC3 and DU145 cells, which contain an unmethylated CpG island in the 14-3-3sigma promoter region. The 14-3-3sigma gene silencing because of CpG methylation correlates with binding of MBD2. In addition, the activation of 14-3-3sigma gene expression by a combination of 5-aza and TSA also involves the release of the MBD2 from the 14-3-3sigma promoter-methylated CpG island in LNCaP and Tramp-C1 cells. Furthermore, MBD2 knockdown by siRNA stimulated 14-3-3sigma expression in LNCaP cells. We also investigated whether the loss of 14-3-3sigma expression in LNCaP and Tramp-C1 cells affects cell proliferation by MTT assays. Interestingly, we observed that 14-3-3sigma-inactivated LNCaP and Tramp-C1 cells had markedly decreased cell proliferation and protein expression of proliferation cell nuclear antigen (PCNA) after restoration of 14-3-3sigma expression with 5-aza and TSA treatment. On the other hand, the same treatment did not significantly affect 14-3-3sigma-active PC3 and DU145 cells, which normally express 14-3-3sigma. Finally, 14-3-3sigma knockdown by siRNA resulted in increased proliferation in PC3 and DU145 cells. These findings suggest that the transcriptional silencing of the 14-3-3sigma gene is caused by promoter CpG island methylation associated with MBD2, and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

14-3-3 Proteins↗
PubMed2006

Negative cell cycle regulator 14-3-3sigma stabilizes p27 Kip1 by inhibiting the activity of PKB/Akt.

The 14-3-3sigma (sigma) protein is a human cancer marker downregulated in various tumors, but its function has not been fully established. 14-3-3sigma is a negative regulator of cell cycle when overexpressed, but it is not clear whether 14-3-3sigma regulates cyclin-dependent kinase inhibitor p27(Kip1) to negatively affect cell cycle progression. Protein kinase B/Akt is a crucial regulator of oncogenic signal and can phosphorylate p27(Kip1) to enhance p27(Kip1)degradation, thereby promoting cell growth. Here, we show that 14-3-3sigma-mediated cell cycle arrest concurred with p27(Kip1) upregulation and Akt inactivation. We show that 14-3-3sigma blocks Akt-mediated acceleration of p27(Kip1) turnover rate. 14-3-3sigma inhibits Akt-mediated p27(Kip1) phosphorylation that targets p27(Kip1) for nuclear export and degradation. 14-3-3sigma inhibits cell survival and tumorigenicity of Akt-activating breast cancer cell. Low expression of 14-3-3sigma in human primary breast cancers correlates with cytoplasmic location of p27(Kip1). These data provide an insight into 14-3-3sigma activity and rational cancer gene therapy by identifying 14-3-3sigma as a positive regulator of p27 and as a potential anticancer agent.

14-3-3 Proteins↗