LIN54 harboring a mutation in CHC domain is localized to the cytoplasm and inhibits cell cycle progression

The mammalian LIN complex (LINC) plays important roles in regulation of cell cycle genes. LIN54 is an essential core subunit of the LINC and has a DNA binding region (CHC domain), which consists of two cysteine-rich (CXC) domains separated by a short spacer. We generated various LIN54 mutants, such as CHC deletion mutant, and investigated their subcellular localizations and effects on cell cycle. Wild-type LIN54 was predominantly localized in the nucleus. We identified two nuclear localization signals (NLSs), both of which were required for nuclear localization of LIN54. Interestingly, deletion of one CXC domain resulted in an increased cytoplasmic localization. The cytoplasmic LIN54 mutant accumulated in the nucleus after leptomycin B treatment, suggesting CRM1-mediated nuclear export of LIN54. Point mutations (C525Y and C611Y) in conserved cysteine residues of CXC domain that abolish DNA binding activity also increased cytoplasmic localization. These data suggest that DNA binding activity of LIN54 is required for its nuclear retention. We also found that LIN54^C525Y and LIN54^C611Y inhibited cell cycle progression and led to abnormal nuclear morphology. Other CXC mutants also induced similar abnormalities in cell cycle progression. LIN54^C525Y led to a decreased expression of some G₂/M genes, whose expressions are regulated by LINC. This cell cycle inhibition was partially restored by overexpression of wild-type LIN54. These results suggest that abnormal cellular localization of LIN54 may have effects on LINC activity.     

A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane

Integral membrane proteins of the inner nuclear membrane (INM) are inserted into the endoplasmic reticulum membrane during their biogenesis and are then targeted to their final destination. We have used human SUN2 to delineate features that are required for INM targeting and have identified multiple elements that collectively contribute to the efficient localization of SUN2 to the nuclear envelope (NE). One such targeting element is a classical nuclear localization signal (cNLS) present in the N‐terminal, nucleoplasmic domain of SUN2. A second motif proximal to the cNLS is a cluster of arginines that serves coatomer‐mediated retrieval of SUN2 from the Golgi. Unexpectedly, also the C‐terminal, lumenal SUN domain of SUN2 supports NE localization, showing that targeting elements are not limited to cytoplasmic or transmembrane domains of INM proteins. Together, SUN2 represents the first mammalian INM protein relying on a functional cNLS, a Golgi retrieval signal and a perinuclear domain to mediate targeting to the INM.     

The circadian mutation PER2(S662G) is linked to cell cycle progression and tumorigenesis

Circadian oscillation and cell cycle progression are the two most essential rhythmic events present in almost all organisms. Circadian rhythms keep track of time and provide temporal regulation with a period of about 24 h. The cell cycle is optimized for growth and division, but not for time keeping. Circadian gated cell divisions are observed in nearly all organisms. However, the implications of this coupling to the physiology of mammals are unknown. A mutation (S662G) in the clock protein PERIOD2 (PER2) is responsible for familial advanced sleep phase syndrome in which sleep onset occurs in the early evening and wakefulness occurs in the early morning. Here, we provide evidence that the PER2^S662 mutation leads to enhanced resistance to X-ray-induced apoptosis and increased E1A- and RAS-mediated oncogenic transformation. Accordingly, the PER2^S662 mutation affects tumorigenesis in cancer-sensitized p53^R172H/+ mice. Finally, analyzing the clock-controlled cell cycle genes p21, c-Myc, Cyclin D1 and p27, we found that the relative phases between p21 and Cyclin D expression profiles have been changed significantly in these Per2 allele mutant mouse embryonic fibroblasts. This key role of the Per2-mediated phase alteration of p21 provides what we believe to be a novel mechanism in understanding cell cycle progression, its plasticity and its resistance to interference.     

LINC00261 elevation inhibits angiogenesis and cell cycle progression of pancreatic cancer cells by upregulating SCP2 via targeting FOXP3

Long non-coding RNAs (lncRNAs) biological functions and molecular mechanisms associated with pancreatic cancer (PC) remain to be poorly elucidated. We aimed to clarify the role of lncRNA LINC00261 (LINC00261) in PC and confirm its regulatory mechanisms. Bioinformatics analysis, RNA pull-down and RIP assays were performed to investigate relationship between LINC00261 and forkhead box P3 (FOXP3). Further, dual-luciferase reporter gene and ChIP assays were employed to confirm the relationship among LINC00261, FOXP3 and sterol carrier protein-2 (SCP2). PC cells were introduced with a series of vectors to verify the effects of LINC00261 and SCP2 on the viability, cell cycle progression, migration and angiogenesis of PC cells. Nude mice with the xenograft tumour were used to evaluate the effects LINC00261 on the tumourigenicity. LINC00261 was lowly expressed in PC tissues and cells. SCP2 was inhibited by LINC00261 through FOXP3. Functionally, upregulated LINC00261 or downregulated SCP2 led to reduced cell viability, migration, angiogenesis and tumourigenicity potentials. This study demonstrated the inhibitory role of LINC00261 in the angiogenesis and cell cycle progression of PC cells. It acts through the negative regulation of SCP2 via targeting FOXP3. Findings in this study highlight a potentially biomarker for PC treatment.     

The nuclear envelope in the plant cell cycle: structure, function and regulation

Background

Higher plants are, like animals, organisms in which successful completion of the cell cycle requires the breakdown and reformation of the nuclear envelope in a highly controlled manner. Interestingly, however, while the structures and processes appear similar, there are remarkable differences in protein composition and function between plants and animals.

Scope

Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events.

Conclusion

Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.     

Multiple intramolecular trafficking signals in RESISTANCE TO POWDERY MILDEW 8.2 are engaged in activation of cell death and defense

The extrahaustorial membrane (EHM) is a host‐derived interfacial membrane encasing the haustorium of powdery mildew fungi. Arabidopsis thaliana RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically targeted to the EHM via two EHM‐targeting signals. Here, we demonstrate that proper coordination between the trafficking forces engaged via the EHM‐targeting signals and the nuclear localization signals (NLSs), as well as the nuclear export signals (NESs), in RPW8.2 is critical for the activation of cell death and defense. We show that in the absence of pathogens, RPW8.2 is partitioned between the cytoplasm and the nucleus, and turned over via both the 26S proteasome‐ and the vacuole‐dependent pathways. Enhanced cytoplasmic localization of RPW8.2 by tagging it with a NES led to lethal cell death. By contrast, enhanced nuclear localization of RPW8.2 by adding an NLS to it resulted in resistance to powdery mildew. Whereas expression of the NES‐containing C‐terminal domain of RPW8.2 in the cytoplasm is sufficient to trigger cell death, no such cell death‐inducing activity is found with RPW8.2 variants that contain the two EHM‐targeting signals along with the NES‐containing C‐terminal domain. In addition, we present evidence for the involvement of a leaf senescence pathway in RPW8.2‐mediated cell death and defense. Taken together, our data suggest that RPW8.2 is subject to adjustment by distinct and perhaps coordinated mechanisms for its localization and function via interaction with the multiple intramolecular trafficking signals, which should provide further insights into RPW8.2‐activated, EHM‐focused resistance against powdery mildew.     

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

LINC00152 promotes cell cycle progression in hepatocellular carcinoma via miR-193a/b-3p/CCND1 axis

Long intergenic non-coding RNA 00152 (LINC00152) is aberrantly expressed in various human malignancies and plays an important role in the pathogenesis. Here, we found that LINC00152 is upregulated in hepatocellular carcinoma (HCC) tissues as compared to adjacent non-neoplastic tissues; gain-and-loss-of-function analyses in vitro showed that LINC00152 facilitates HCC cell cycle progression through regulating the expression of CCND1. LINC00152 knockdown inhibits tumorigenesis in vivo. MS2-RIP analysis indicated that LINC00152 binds directly to miR-193a/b-3p, as confirmed by luciferase reporter assays. Furthermore, ectopic expression of LINC00152 partially halted the decrease in CCND1 expression and cell proliferation capacity induced by miR-193a/b-3p overexpression. Thus, LINC00152 acts as a competing endogenous RNA (ceRNA) by sponging miR-193a/b-3p to modulate its target gene, CCND1. Our findings establish a ceRNA mechanism regulating cell proliferation in HCC via the LINC00152/miR-193a/b-3p/CCND1 signalling axis, and identify LINC00152 as a potential therapeutic target for HCC.     

Transmembrane domain is crucial to the subcellular localization and function of Myc target 1

Deregulation of c‐MYC occurs in a variety of human cancers. Overexpression of c‐MYC promotes cell growth, proliferation, apoptosis, transformation and genomic instability. MYC target 1 (MYCT1) is a direct target gene of c‐MYC, and its murine homologue MT‐MC1 recapitulated multiple c‐Myc‐related phenotypes. However, the molecular mechanism of MYCT1 remains unclear. Here, we identified the transmembrane (TM) domain of MYCT1, not the nuclear localization sequence, is indispensable to the vesicle‐associated localization of MYCT1 protein in the cytoplasmic membrane vesicle. Overexpression of MYCT1, not MYCT1 (ΔTM), decreased cell viability under serum deprivation and increased tumour cell migration ability. We further identified CKAP4 interacted with MYCT1 and contributed to the function of MYCT1. In addition, we found that a mutation, A88D, which is observed in patient sample, changed the localization, and abolished the effect on cell viability and cell migration, suggesting that the TM domain is critical to MYCT1.     

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation.     

Phospholipase Cdelta1 associates with importin beta1 and translocates into the nucleus in a Ca2+-dependent manner

Phospholipase C (PLC)delta1 shuttles between the nucleus and the cytoplasm. Here, we demonstrate that treatment of MDCK cells and PC12 cells with ionomycin causes nuclear accumulation of ectopically expressed and endogenous PLCdelta1, respectively, suggesting that signals that increase [Ca2+]i trigger nuclear translocation. To clarify the molecular mechanisms involved in this translocation, we have examined whether PLCdelta1 binds with importins. PLCdelta1 interacted with importin beta1 in a Ca2+-dependent manner in vitro even in the absence of importin alpha. A PLCdelta1 mutant E341A, which lacks Ca2+-binding to the catalytic core, did not show this interaction at any physiological Ca2+ concentration and did not translocate into the nucleus after ionomycin treatment when expressed in MDCK cells. These results suggested that the nuclear import of PLCdelta1 is mediated by its Ca2+-dependent interaction with importin beta1.     

Acidic domain is indispensable for MDM2 to negatively regulate the acetylation of p53

MDM2 is the most important negative regulator of tumor suppressor p53. Both RING finger domain and acidic domain of MDM2 contribute to the ubiquitination of p53. The crosstalk between ubiquitination and acetylation of p53 prompts us to examine whether acidic domain is essential for MDM2 to regulate the acetylation of p53. We find that the acidic domain of MDM2 is necessary to inhibit p300-mediated acetylation of p53 as well as to mediate the deacetylation of p53. Our results indicate that acidic domain of MDM2 provides essential information for acetyltransferase p300 and deacetylase HDAC1 and is indispensable for MDM2 to negatively regulate the acetylation of p53.     

The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.     

C-terminal domain of p16(INK4a) is adequate in inducing cell cycle arrest, growth inhibition and CDK4/6 interaction similar to the full length protein in HT-1080 fibrosarcoma cells

The tumor suppressor p16(INK4a) has earned widespread attention in cancer studies since its discovery as an inhibitor of cyclin-dependent kinases (CDKs) 4/6. Structurally, it consists of four complete ankyrin repeats, believed to be involved in CDK4 interaction. According to the previous disparities concerning the importance of domains and inactivating mutations in p16, we aimed to search for the domain possessing the functional properties of the full length protein. Upon our in silico screening analyses followed by experimental assessments, we have identified the novel minimum functional domain of p16 to be the C-terminal half including ankyrin repeats III, IV and the C-terminal flanking region accompanied by loops 2 and 3. Transfection of this truncated form into HT-1080 human fibrosarcoma cells, lacking endogenous p16, revealed that it is able to inhibit cell growth and proliferation equivalent to p16(INK4a). The functional analysis showed that this fragment like p16 can interact with CDK4/6, block the entry into S phase of the cell cycle and suppress growth as indicated by colony formation assay. Identification of p16 minimum functional domain can be of benefit to the future peptidomimetic drug design as well as gene transfer for cancer therapy.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.