Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Cell cycle-dependent modulations of fenestrin expression in Tetrahymena pyriformis

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycle-regulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila.     

Cell cycle-dependent changes in localization of a 210-kDa microtubule-interacting protein in Leishmania

Using the monoclonal antibody MA-01, a new 210-kDa microtubule-interacting protein was identified in Leishmania promastigotes by immunoblotting and by immunoprecipitation. The protein was thermostable and was located on microtubules prepared by taxol-driven polymerization in vitro. On fixed cells the antibody gave specific staining of flagellum, flagellar pocket, and mitotic spindle. Subpellicular microtubules were basically not decorated but posterior poles of the cells were labeled in a cell-cycle-dependent manner. In anterior and posterior poles of cells the 210-kDa protein codistributed with the 57-kDa protein, immunodetected with anti-vimentin antibody, that was located only on cell poles. Immunolocalization of the 57-kDa protein was most prominent in dividing cells. The presented data suggest that the 210-kDa protein is a newly identified microtubule-interacting protein of Leishmania that could be involved in anchoring the microtubules in posterior poles of these cells. The striking codistribution of the microtubule-interacting protein and the 57-kDa protein in protozoa is described for the first time.     

Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts.

The purpose of this investigation is to determine whether the levels of cyclooxygenase-2 (COX-2) expression are cell cycle dependent. We used a serum-starved human foreskin fibroblast model to determine changes in COX-2 mRNA, protein, and promoter activity in response to stimulation with interleukin-1b (IL-1b) and phorbol 12-myristate 13-acetate (PMA) at G0, G1, S and G2/M phases of the cell cycle. IL-1b (1 ng/ml) and PMA (100 nM) induced robust COX-2 expression in the G0 cells, and the level of COX-2 expression declined progressively after the cells had entered the cell cycle. The COX-2 mRNA level at G1, S and G2/M phases of the cell cycle was 76%, 46%, and 30% of that at G0, respectively. A 5-flanking promoter fragment of COX-2 constructed into a luciferase expression vector was transfected into cells. The promoter activity in response to PMA stimulation was significantly higher in G0 than in S phase cells. These results imply that G0 cells are the key players in inflammation and other COX-2-dependent pathophysiological processes. When the cells are in the proliferative phase, COX-2 inducibility becomes restrained probably by an endogenous control mechanism to avoid COX-2 mediated oxidative DNA damage.     

Cell cycle-dependent expression and subcellular localization of fructose 1,6-bisphosphatase

Recently a gluconeogenic enzyme was discovered—fructose 1,6-bisphosphatase (FBPase)—that localizes in the nucleus of a proliferating cell, but its physiological role in this compartment remains unclear. Here, we demonstrate the link between nuclear localization of FBPase and the cell cycle progression. Results of our studies indicate that in human and mouse squamous cell lung cancer, as well as in the HL-1 cardiomyocytes, FBPase nuclear localization correlates with nuclear localization of S and G2 phase cyclins. Additionally, activity and expression of the enzyme depends on cell cycle stages. Identification of FBPase interacting partners with mass spectrometry reveals a set of nuclear proteins involved in cell cycle regulation, mRNA processing and in stabilization of genomic DNA structure. To our knowledge, this is the first experimental evidence that muscle FBPase is involved in cell cycle events.     

Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression

Intracellular microbes have evolved efficient strategies for transitioning from one cell to another in a process termed intercellular transmission. Here we show that host cell transmission of the obligate intracellular parasite Toxoplasma gondii is closely tied to specific cell cycle distributions, with egress and reinvasion occurring most proficiently by parasites in the G1 phase. We also reveal that Toxoplasma undergoes marked changes in mRNA expression when transitioning from the extracellular environment to its intracellular niche. These mRNA level changes reflect a modal switch from expression of proteins involved in invasion, motility and signal transduction in extracellular parasites to expression of metabolic and DNA replication proteins in intracellular parasites. Host cell binding and signalling associated with the discharge of parasite secretory proteins was not sufficient to induce this switch in gene expression, suggesting that the regulatory mechanisms responsible are tied to the establishment of the intracellular environment. The genes whose expression increased after parasite invasion belong to a progressive cascade known to underlie the parasite division cycle indicating that the unique relationship between the G1 phase and invasion effectively synchronizes short-term population growth. This work provides new insight into how this highly successful parasite competently transits from cell to cell.     

Cell cycle-dependent binding of HMGN proteins to chromatin

Throughout the cell cycle, the histones remain associated with DNA, but the repertoire of proteins associated with the chromatin fiber continuously changes. The chromatin interaction of HMGNs, a family of nucleosome binding proteins that modulates the structure and activity of chromatin, during the cell cycle is controversial. Immunofluorescence studies demonstrated that HMGNs are not associated with chromatin, whereas live cell imaging indicated that they are present in mitotic chromosomes.     

Cell cycle-dependent expression of Kv1.5 is involved in myoblast proliferation

Voltage-dependent K(+) channels (Kv) are involved in the proliferation of many types of cells, but the mechanisms by which their activity is related to cell growth remain unclear. Kv antagonists inhibit the proliferation of mammalian cells, which is of physiological relevance in skeletal muscle. Although myofibres are terminally differentiated, some resident myoblasts may re-enter the cell cycle and proliferate. Here we report that the expression of Kv1.5 is cell-cycle dependent during myoblast proliferation. In addition to Kv1.5 other Kv, such as Kv1.3, are also up-regulated. However, pharmacological evidence mainly implicates Kv1.5 in myoblast growth. Thus, the presence of S0100176, a Kv antagonist, but not margatoxin and dendrotoxin, led to cell cycle arrest during the G(1)-phase. The use of selective cell cycle blockers showed that Kv1.5 was transiently accumulated during the early G(1)-phase. Furthermore, while myoblasts treated with S0100176 expressed low levels of cyclin A and D(1), the expression of p21(cip-1) and p27(kip1), two cyclin-dependent kinase inhibitors, increased. Our results indicate that the cell cycle-dependent expression of Kv1.5 is involved in skeletal muscle cell proliferation.     

Cell cycle-dependent surface changes in Chinese hamster cells grown in suspension culture

The cell surface appears to play an important part in the control of cell replication. It has been demonstrated that the cell membrane undergoes cyclic changes in appearance which bear a relation to the cell cycle phase, irrespective of close intercellular contact. The surface of Chinese hamster (CHO) cells was investigated using the scanning electron microscope (SEM). The cells were synchronized in suspension culture and were sampled at frequent intervals during the cell cycle. During mitosis, the cells showed microvilli and few blebs. In early G 1 phase, profuse microvilli were seen. In late G 1 phase, blebs appeared and persisted in great numbers. During the synthesis of DNA in the S phase, blebs were observed in the early stages and then declined in number; in G 2 phase, the blebs appeared to be larger (1–2 μm) and more sparsely distributed than in late S phase. Some of these blebs were pedunculated and, in some instances, the diameter of the pedicles approximated the diameter of microvilli. Since the reasons for these changes are not understood, our long-range goal is to correlate the observed surface changes with internal biochemical events during the cell cycle.     

Csk,a critical link of g protein signals to actin cytoskeletal reorganization

Heterotrimeric G proteins can signal to reorganize the actin cytoskeleton, but the mechanism is unclear. Here we report that, in tyrosine kinase Csk-deficient mouse embryonic fibroblast cells, G protein (Gbetagamma, Galpha(12), Galpha(13), and Galpha(q))-induced, and G protein-coupled receptor-induced, actin stress fiber formation was completely blocked. Reintroduction of Csk into Csk-deficent cells restored the G protein-induced actin stress fiber formation. Chemical rescue experiments with catalytic mutants of Csk demonstrated that the catalytic activity of Csk was required for this process. Furthermore, we uncovered that Gbetagamma can both translocate Csk to the plasma membrane and directly increase Csk kinase activity. Our genetic and biochemical studies demonstrate that Csk plays a critical role in mediating G protein signals to actin cytoskeletal reorganization.     

Cell cycle-dependent assembly of a Gin4-septin complex

Gin4, a Nim1-related kinase, is required in budding yeast for localization of the septins and for proper control of daughter cell growth during G2/M. Gin4 becomes hyperphosphorylated when cells enter mitosis, leading to activation of Gin4 kinase activity. In this study, we have used immunoaffinity chromatography to identify proteins that associate with Gin4 during mitosis, with the goal of finding targets of Gin4 kinase activity and proteins that play a role in Gin4 activation. We show that during mitosis Gin4 is assembled into a multiprotein complex that includes Nap1, Bni5, the septins, and at least two molecules of Gin4. The associated Gin4 molecules present in this complex phosphorylate each other, leading to Gin4 hyperphosphorylation. Furthermore, the Shs1 septin present in the complex undergoes Gin4-dependent phosphorylation during mitosis and appears to be a substrate of Gin4 in vitro, suggesting that it is a target of Gin4 kinase activity in vivo. Genetic data support the idea that Shs1 is an important target of Gin4 kinase activity. Association of Gin4 with the septins during mitosis requires Shs1, Nap1, Cla4, Elm1, and the kinase activities of Gin4 and Cdc28. Self-association of Gin4 molecules requires Shs1 but not Cla4 or Nap1. Previous work has suggested that the septins function together as a tight complex, and we found that the majority of the Shs1 in the cell is tightly bound to the other septins Cdc3, Cdc10, Cdc11, and Cdc12. Interestingly, however, Shs1 can bind to Gin4 and induce Gin4 oligomerization under conditions in which the Cdc11 septin does not bind to Gin4, suggesting that Shs1 can function independently of the other septins. Taken together, these findings suggest that highly regulated protein-binding events ensure that the Gin4 kinase is activated only during mitosis and only in association with Shs1, a likely in vivo substrate of Gin4. In addition, these results provide clues to how Gin4 may regulate the localization or function of the septins.     

Cell cycle-dependent changes in tissue transglutaminase mRNA levels in bovine endothelial cells.

The pattern of transglutaminase gene expression through the cell cycle was examined by Northern blot analysis using cultured bovine endothelial cells and a cDNA probe. When the cells reached confluency or were arrested in G0/G1 phase by nutrition deprivation, transglutaminase mRNA rose to a very high level; S- and M-phase extracts showed high and low levels, respectively. Subcellular localization studies by sucrose gradient centrifugation and immunostaining demonstrated that the majority of transglutaminase is present in cytosols throughout the cycle. The cell cycle-dependent changes in the transglutaminase mRNA levels strongly support the implicated involvement of the enzyme in cell growth, differentiation, and senescence.