DEPDC1 is a novel cell cycle related gene that regulates mitotic progression

DEPDC1 is a recently identified novel tumor-related gene that is upregulated in several types of cancer and contributes to tumorigenesis. In this study, we have investigated the expression pattern and functional implications of DEPDC1 during cell cycle progression. Expression studies using synchronized cells demonstrated that DEPDC1 is highly expressed in the mitotic phase of the cell cycle. Immunofluorescence assays showed that DEPDC1 is predominantly localized in the nucleus during interphase and is redistributed into the whole cell upon nuclear membrane breakdown in metaphase. Subsequently, siRNA-mediated knockdown of DEPDC1 caused a significant mitotic arrest. Moreover, knockdown of DEPDC1 resulted in remarkable mitotic defects such as abnormal multiple nuclei and multipolar spindle structures accompanied by the upregulation of the A20 gene as well as several cell cycle-related genes such as CCNB1 and CCNB2. Taken together, our current observations strongly suggest that this novel cancerous gene, DEPDC1, plays a pivotal role in the regulation of proper mitotic progression. [BMB Reports 2015; 48(7): 413-418]     

Evidence that the phosphatidylinositol cycle is linked to cell motility

Transmembrane signaling via specific ligand/receptor interactions induces the immediate polymerization of actin and formation of microfilament assemblies close to the plasma membrane. The profilin:actin complex appears to provide the actin for this filament formation. A clue to the nature of the regulatory mechanism involved was recently found in that phosphatidylinositol 4,5-bisphosphate can bind to profilin, dissociate the profilactin complex, and thus liberate actin for polymerization. This suggests that the phosphatidylinositol (PI) cycle, which plays important roles in cellular regulation, also might control microfilament-based motility. We show here that neomycin, a drug which has a high affinity for phosphoinositides and in vivo interferes with the PI cycle, inhibits the polymerization of actin in platelets induced either by thrombin or by ADP. When ADP was used as agonist (but not in the case of thrombin) the induction of actin polymerization could also be blocked by the addition of aspirin. Introduction of Ca2+ into platelets by the use of the ionophore A23187 or stimulation of protein kinase C (PkC) by the phorbol ester TPA did not induce actin polymerization; neither did the addition of a combination of these two agents. Retinoic acid which inhibits PkC was also without effect on thrombin-induced actin polymerization.     

Ubiquitin/proteasome-dependent degradation of D-type cyclins is linked to tumor necrosis factor-induced cell cycle arrest

Tumor necrosis factor-alpha (TNF) is well known for its cytotoxic effect on malignant cells. Its role in cell cycle control is relatively less known. In this study, we found that TNF induced G(1) arrest of TF-1 and MV4-11 cells while simultaneously causing apoptosis. Treatment of the cells with TNF for 48 h caused cell cycle arrest, accompanied by dephosphorylation of pRb and reduction in D-type cyclin expression. The down-regulation of the D-type cyclins resulted in approximately 50-80% decrease of the cyclin-dependent kinase activities. Cells treated with calpain-dependent inhibitor ALLN and apoptosis inhibitor zVAD-FMK suppressed degradation of IkappaBalpha and activation of caspase 3, respectively. However, treatment of cells with these two inhibitors was not able to prevent TNF-induced down-regulation of the D-type cyclins. In contrast, proteasome inhibitor MG-132 and lactacystin blocked both TNF-induced degradation of IkappaBalpha and down-regulation of D-type cyclins. These data suggest that down-regulation of D-type cyclins by TNF may be proteasome-proteolysis dependent. Additional support for this conclusion was obtained from experiments showing an increase of proteasome activity in TNF-treated cells and in vitro degradation of cyclin D3 by 26 S proteasome.     

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.     

Spindle assembly checkpoint regulates mitotic cell cycle progression during preimplantation embryo development

Errors in chromosome segregation or distribution may result in aneuploid embryo formation, which causes implantation failure, spontaneous abortion, genetic diseases, or embryo death. Embryonic aneuploidy occurs when chromosome aberrations are present in gametes or early embryos. To date, it is still unclear whether the spindle assembly checkpoint (SAC) is required for the regulation of mitotic cell cycle progression to ensure mitotic fidelity during preimplantation development. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of SAC components (Bub3, BubR1 and Mad2) in mouse preimplantation embryos. Our data showed that overexpressed SAC components inhibited metaphase-anaphase transition by preventing sister chromatid segregation. Deletion of SAC components by RNAi accelerated the metaphase-anaphase transition during the first cleavage and caused micronuclei formation, chromosome misalignment and aneuploidy, which caused decreased implantation and delayed development. Furthermore, in the presence of the spindle-depolymerizing drug nocodazole, SAC depleted embryos failed to arrest at metaphase. Our results suggest that SAC is essential for the regulation of mitotic cell cycle progression in cleavage stage mouse embryos.     

Targeted degradation of the cyclin-dependent kinase inhibitor ICK4/KRP6 by RING-type E3 ligases is essential for mitotic cell cycle progression during Arabidopsis gametogenesis

Following meiosis, plant gametophytes develop through two or three rounds of mitosis. Although the ontogeny of gametophyte development has been defined in Arabidopsis thaliana, the molecular mechanisms regulating mitotic cell cycle progression are not well understood. Here, we report that RING-H2 group F 1a (RHF1a) and RHF2a, two RING-finger E3 ligases, play an important role in Arabidopsis gametogenesis. The rhf1a rhf2a double mutants are defective in the formation of male and female gametophytes due to interphase arrest of the mitotic cell cycle at the microspore stage of pollen development and at female gametophyte stage 1 of embryo sac development. We demonstrate that RHF1a directly interacts with and targets a cyclin-dependent kinase inhibitor ICK4/KRP6 (for Interactors of Cdc2 Kinase 4/Kip-related protein 6) for proteasome-mediated degradation. Inactivation of the two redundant RHF genes leads to the accumulation of ICK4/KRP6, and reduction of ICK4/KRP6 expression largely rescues the gametophytic defects in rhf1a rhf2a double mutants, indicating that ICK4/KRP6 is a substrate of the RHF E3 ligases. Interestingly, in situ hybridization showed that ICK4/KRP6 was predominantly expressed in sporophytes during meiosis. Our findings indicate that RHF1a/2a-mediated degradation of the meiosis-accumulated ICK4/KRP6 is essential to ensure the progression of subsequent mitoses to form gametophytes in Arabidopsis.     

Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity.

Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is down-regulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21(WAF/CIP)-dependent manner. To understand how mitogen-induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen-activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21(WAF/CIP) nor the tumor suppressor p53 was required for Gax-induced cell death. Arrest in G1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax-mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl-2 down-regulation and Bax up-regulation in mitogen-activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax-induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen-dependent pathways that circumvent the cell cycle to alter Bcl-2 family protein expression.     

The Drosophila gene taranis encodes a novel trithorax group member potentially linked to the cell cycle regulatory apparatus

Genes of the Drosophila Polycomb and trithorax groups (PcG and trxG, respectively) influence gene expression by modulating chromatin structure. Segmental expression of homeotic loci (HOM) initiated in early embryogenesis is maintained by a balance of antagonistic PcG (repressor) and trxG (activator) activities. Here we identify a novel trxG family member, taranis (tara), on the basis of the following criteria: (i) tara loss-of-function mutations act as genetic antagonists of the PcG genes Polycomb and polyhomeotic and (ii) they enhance the phenotypic effects of mutations in the trxG genes trithorax (trx), brahma (brm), and osa. In addition, reduced tara activity can mimic homeotic loss-of-function phenotypes, as is often the case for trxG genes. tara encodes two closely related 96-kD protein isoforms (TARA-alpha/-beta) derived from broadly expressed alternative promoters. Genetic and phenotypic rescue experiments indicate that the TARA-alpha/-beta proteins are functionally redundant. The TARA proteins share evolutionarily conserved motifs with several recently characterized mammalian nuclear proteins, including the cyclin-dependent kinase regulator TRIP-Br1/p34(SEI-1), the related protein TRIP-Br2/Y127, and RBT1, a partner of replication protein A. These data raise the possibility that TARA-alpha/-beta play a role in integrating chromatin structure with cell cycle regulation.     

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.     

An essential cell cycle-regulated nucleolar protein relocates to the mitotic spindle where it is involved in mitotic progression in Trypanosoma brucei

TbNOP86 and TbNOP66 are two novel nucleolar proteins isolated in Trypanosoma brucei. They share 92.6% identity, except for an additional C-terminal domain of TbNOP86 of 182 amino acids in length. Both proteins are found in Trypanosomatidae, but similarity to other eukaryotic proteins could not be found. TbNOP86 and TbNOP66 are expressed at similar level in procyclic and bloodstream forms, although the relative level of expression of TbNOP66 is 11 times lower. TbNOP86 undergoes post-translational modifications, as it is found predominantly at 110 kDa compared with the predicted 86 kDa. Immunofluorescence of overexpressed ty-tagged TbNOP86 and TbNOP66 showed that both proteins accumulated in the nucleolus of G(1) cells. This was confirmed by the co-localization of an endogenous TbNOP86-myc with the nucleolar protein Nopp140. TbNOP86-ty localization is cell cycle-regulated, because it colocalizes with the mitotic spindle in mitotic cells. TbNOP86 is required for mitotic progression in both life stages as depleted cells are enriched in the G(2)/M phase. In procyclic cells, a reduced growth rate is accompanied by an accumulation of zoids (0N1K), 2N1K, and multinucleated cells (xNyK). The 2N1K cells are blocked in late mitosis as nucleolar segregation is completed. TbNOP86 depletion in bloodstream form caused a drastic growth inhibition producing cells bearing two kinetoplasts and an enlarged nucleus (1N(*)2K), followed by an accumulation of 2N2K cells with connected nuclei and xNyK cells. These studies of TbNOP86 provide a more comprehensive account of proteins involved in mitotic events in trypanosomes and should lead to the identification of partners with similar function.     

Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression

Hsp70 proteins represent a family of chaperones that regulate cellular homeostasis and are required for cancer cell survival. However, their function and regulation in mitosis remain unknown. In this paper, we show that the major inducible cytoplasmic Hsp70 isoform, Hsp72, is required for assembly of a robust bipolar spindle capable of efficient chromosome congression. Mechanistically, Hsp72 associates with the K-fiber–stabilizing proteins, ch-TOG and TACC3, and promotes their interaction with each other and recruitment to spindle microtubules (MTs). Targeting of Hsp72 to the mitotic spindle is dependent on phosphorylation at Thr-66 within its nucleotide-binding domain by the Nek6 kinase. Phosphorylated Hsp72 concentrates on spindle poles and sites of MT–kinetochore attachment. A phosphomimetic Hsp72 mutant rescued defects in K-fiber assembly, ch-TOG/TACC3 recruitment and mitotic progression that also resulted from Nek6 depletion. We therefore propose that Nek6 facilitates association of Hsp72 with the mitotic spindle, where it promotes stable K-fiber assembly through recruitment of the ch-TOG–TACC3 complex.     

Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint

Some cells have a quality control checkpoint that can detect a single misattached chromosome and delay the onset of anaphase, thus allowing time for error correction. The mechanical error in attachment must somehow be linked to the chemical regulation of cell cycle progression. The 3F3 antibody detects phosphorylated kinetochore proteins that might serve as the required link (Gorbsky, G. J., and W. A. Ricketts. 1993. J. Cell Biol. 122:1311-1321). We show by direct micromanipulation experiments that tension alters the phosphorylation of kinetochore proteins. Tension, whether from a micromanipulation needle or from normal mitotic forces, causes dephosphorylation of the kinetochore proteins recognized by 3F3. If tension is absent, either naturally or as a result of chromosome detachment by micromanipulation, the proteins are phosphorylated. Equally direct experiments identify tension as the checkpoint signal: tension from a microneedle on a misattached chromosome leads to anaphase (Li, X., and R. B. Nicklas. 1995. Nature (Lond.). 373:630-632), and we show here that the absence of tension caused by detaching chromosomes from the spindle delays anaphase indefinitely. Thus, the absence of tension is linked to both kinetochore phosphorylation and delayed anaphase onset. We propose that the kinetochore protein dephosphorylation caused by tension is the all clear signal to the checkpoint. The evidence is circumstantial but rich. In any event, tension alters kinetochore chemistry. Very likely, tension affects chemistry directly, by altering the conformation of a tension-sensitive protein, which leads directly to dephosphorylation.     

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.     

TBX5 is required for embryonic cardiac cell cycle progression

Despite the critical importance of TBX5 in normal development and disease, relatively little is known about the mechanisms by which TBX5 functions in the embryonic heart. Our present studies demonstrate that TBX5 is necessary to control the length of the embryonic cardiac cell cycle, with depletion of TBX5 leading to cardiac cell cycle arrest in late G(1)- or early S-phase. Blocking cell cycle progression by TBX5 depletion leads to a decrease in cardiac cell number, an alteration in the timing of the cardiac differentiation program, defects in cardiac sarcomere formation, and ultimately, to cardiac programmed cell death. In these studies we have also established that terminally differentiated cardiomyocytes retain the capacity to undergo cell division. We further show that TBX5 is sufficient to determine the length of the embryonic cardiac cell cycle and the timing of the cardiac differentiation program. Thus, these studies establish a role for TBX5 in regulating the progression of the cardiac cell cycle.     

Commitment to the mitotic cell cycle in yeast in relation to meiosis

Under restrictive vegetative conditions, cells of cell-division cycle (cdc) temperature-sensitive mutants arrest at specific points in the cycle. Meiotic and mitotic behaviour of such arrested cells was examined under permissive sporulation conditions. Those mutants which were committed to mitosis at their specific point of arrest finished the cell cycle and could only then go into meiosis. It was found that commitment to mitosis occurred early in the cell cycle, prior to DNA replication, and that this commitment was dependent upon the gene function of cdc4.     

RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation

RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.     

Vitamin D receptor expression is linked to cell cycle control in normal human keratinocytes

To improve our understanding of the cutaneous vitamin D system, we studied vitamin D receptor (VDR) gene regulation in cultured human keratinocytes. Because VDR and its ligand 1 alpha,25-dihydroxyvitamin D(3) have been implicated in epidermal growth control, we investigated VDR expression as related to cellular proliferation by using different cell cycle synchronization protocols. Keratinocytes, deprived of growth factors, were forced into quiescence and a concomitant loss of VDR expression was observed. Mitogenic stimulation of these G(0) cells however quickly upregulated VDR levels several hours ahead the G(1)-S transition point. Growth arrest at the G(1)-S border by mimosine treatment or at the metaphase by nocodazole also downregulated VDR levels but a restoration of VDR expression was again quickly achieved after reentering the cell cycle. These findings indicate that VDR expression in keratinocytes is restricted to actively cycling cells, but not limited to one particular phase of the cell cycle.