Calmodulin and cell cycle control.

Previous studies have indicated a role for the calcium receptor calmodulin in the control of eukaryotic cell proliferation. Using a molecular genetic approach in the filamentous fungus Aspergillus nidulans we have shown that CaM is required for cell cycle progression at multiple points in the cell cycle. Construction of an A nidulans strain conditional for calmodulin expression reveals that this protein is required during G1/S and for the initiation of mitosis. A lack of calmodulin results in cell cycle arrest, and a failure in polar growth that accompanies germination of A nidulans spores. In addition, increased expression of calmodulin in this organism permits growth at suboptimal calcium concentrations, indicating that cell growth is coordinately regulated by calcium and calmodulin. Together these results indicate that calmodulin-dependent processes may be conserved between A nidulans and vertebrate cells, and suggest that this approach may allow us to elucidate the molecular mechanism underlying calmodulin-regulated control of cell proliferation.     

Calmodulin is required for cell-cycle progression during G1 and mitosis.

In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine-papilloma-virus (BPV)-based expression vectors capable of inducibly synthesizing CaM sense (BPV-MCM) or anti-sense (BPV-CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV-MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV-CaMAS vector transiently produce CaM anti-sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti-sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell-cycle progression under normal conditions of growth.     

Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle

Ca2+ ions intervene during different phases of the progression of the cell cycle, but only one calcium-binding protein, calmodulin, has been shown to be associated with dividing cells. We therefore screened cancer cells for the presence of other related calcium-binding proteins. Using molecular biological and immunohistochemical techniques we show that human tumor cells of epithelial origin, express calretinin. Calretinin immunoreactivity can be demonstrated at precise moments of the cell cycle and, in particular, in phase G1 and during mitosis. During mitosis calretinin is localized both in the cytoplasm and in the mitotic spindle. In the cytoplasm we find calretinin after prophase and until telophase. In the spindle apparatus, calretinin is already present in cells in prometaphase and persists in all the succeeding mitotic phases. It is associated with the kinetochore microtubules but, in contrast to calmodulin, also with the polar microtubules. The role that calretinin plays in well-defined moments of the cell cycle of these cells is as yet unknown, but our results strongly suggest that, in collaboration with other molecules, calretinin intervenes in the dynamic phenomena regulating the separation of the chromosomes.     

钙调素对细胞周期的调节

RC3细胞是一种用真核表达载体1~(CaM)转染NIH 3T3细胞建成的可调钙凋素(Calmodulin,CaM)高表达细胞模型。通过分子杂交及蛋白免疫印迹方法证实在地塞米松(Dexamethasome,DXM)作用下,RC3细胞可高表达CaM。CaM的过表达使G_1期细胞减少,S期细胞增加;CaM拮抗剂三氟拉嗪(trifluoperazine,TFP)则使G_1期细胞增加,S期细胞减少。高表达CaM使细胞分裂指数提高,G_2期细胞减少,有丝分裂前期细胞增加,M中期细胞比例下降。而TFP处理则使分裂指数下降,G_2期细胞增加,M前期细胞减少,M中期细胞增加。实验结果表明CaM在G_1/S、G_2/M和M中期/M后期3个位点上对细胞周期进行调控;通过加速G_1至S期,G_2至M期和M中期至M后期的进程,使细胞倍增时间缩短,促进细胞增殖。本工作表明,RC3细胞作为CaM表达可调细胞模型,是研究细胞周期调控的有力工具。     

Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells

The mixed-lineage kinases (MLK) are serine/threonine protein kinases that regulate mitogen-activated protein (MAP) kinase signaling pathways in response to extracellular signals. Recent studies indicate that MLK activity may promote neuronal cell death through activation of the c-Jun NH2-terminal kinase (JNK) family of MAP kinases. Thus, inhibitors of MLK activity may be clinically useful for delaying the progression of neurodegenerative diseases, such as Parkinson's. In proliferating non-neuronal cells, MLK may have the opposite effect of promoting cell proliferation. In the current studies we examined the requirement for MLK proteins in regulating cell proliferation by examining MLK function during G2 and M-phase of the cell cycle. The MLK inhibitor CEP-11004 prevented HeLa cell proliferation by delaying mitotic progression. Closer examination revealed that HeLa cells treated with CEP-11004 during G2-phase entered mitosis similar to untreated G2-phase cells. However, CEP-11004 treated cells failed to properly exit mitosis and arrested in a pro-metaphase state. Partial reversal of the CEP-11004 induced mitotic arrest could be achieved by overexpression of exogenous MLK3. The effects of CEP-11004 treatment on mitotic events included the inhibition of histone H3 phosphorylation during prophase and prior to nuclear envelope breakdown and the formation of aberrant mitotic spindles. These data indicate that MLK3 might be a unique target to selectively inhibit transformed cell proliferation by disrupting mitotic spindle formation resulting in mitotic arrest.     

Cell cycle-dependent regulation of store-operated I(CRAC) and Mg2+-nucleotide-regulated MagNuM (TRPM7) currents

Calcium signaling is a central mechanism for numerous cellular functions and particularly relevant for immune cell proliferation. However, the role of calcium influx in mitotic cell cycle progression is largely unknown. We here report that proliferating rat mast cells RBL-2H3 tightly control their major store-operated calcium influx pathway, I(CRAC), during cell cycle progression. While I(CRAC) is maintained at control levels during the first gap phase (G1), the current is significantly up-regulated in preparation for and during chromatin duplication. However, mitosis strongly suppresses I(CRAC). Non-proliferating cells deprived of growth hormones strongly down-regulate I(CRAC) while increasing cell volume. We further show that the other known calcium (and magnesium) influx pathway in mast cells, the TRPM7-like magnesium-nucleotide-regulated metal (MagNuM) current, is largely uncoupled from cell cycle regulation except in G1. Taken together, our results demonstrate that both store-operated calcium influx via I(CRAC) and MagNuM are regulated at crucial checkpoints during cell cycle progression.     

Ca(2+)/calmodulin-dependent kinase is essential for both growth and nuclear division in Aspergillus nidulans.

The calmodulin gene has been shown to be essential for cell cycle progression in a number of eukaryotic organisms. In vertebrates and Aspergillus nidulans the calmodulin dependence also requires calcium. We demonstrate that the unique gene encoding a multifunctional calcium/calmodulin-dependent protein kinase (CaMK) is also essential in A. nidulans. This enzyme is required both for the nuclear division cycle and for hyphal growth, because spores containing the disrupted gene arrest with a single nucleus and fail to extend a germ tube. A strain conditional for the expression of CaMK was created. When grown under conditions that resulted in a 90% decrease in the enzyme, both nuclear division and growth were markedly slowed. The CaMK seems to be important for progression from G2 to mitosis.     

Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.     

Calmodulin is involved in regulation of cell proliferation.

A chicken calmodulin (CaM) gene has been expressed in mouse C127 cells using a bovine papilloma virus (BPV)-based vector (BPV-CM). The vector-borne genes produce a mature mRNA of the expected size that is present on cytoplasmic polyribosomes. In clonal cell lines transformed by BPV-CM, expression of the CaM gene produced CaM levels 2- to 4-fold above those observed in cells transformed by BPV alone. Increased intracellular CaM caused a reduction of cell cycle length that is solely due to a reduction in the length of the G1 phase. A comparison of six cell lines revealed a linear relationship between the intracellular CaM concentration and the rate of G1 progression. These data provide the first evidence that specific elevation of CaM levels directly affects the rate of cell proliferation.     

Effect of serine/threonine protein kinases and protein phosphatases inhibitors on mitosis progression in a synchronized tobacco BY-2 culture

In order to investigate the role of various serine/threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells, the influence of cyclin(olomoucine) and Ca²⁺/calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine), and a protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin-dependent protein kinases and protein kinase C caused a prophase delay, reduced the mitotic index, and displaced the mitotic peak as compared with control cells. Inhibition of Ca²⁺/calmodulin-dependent protein kinases enhanced the cells entry into prophase and delayed their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances synchronized BY-2 cells entering into all phases of mitosis.     

Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: a novel method suitable for examining dynamic changes in proteins during mitotic progression

Mitosis is a continuous process to separate replicated chromosomes into two daughter cells through prophase, metaphase, anaphase, and telophase. Although a number of methods have been established to synchronize cells at different phases of the cell cycle, it is difficult to synchronize cells at the specific phases, anaphase and telophase, during mitosis because of the short duration of anaphase. Here, we show that HeLa S3 cells in anaphase and in telophase are successfully enriched by treatment with a combination of low concentrations of the microtubule-depolymerizing agent nocodazole and the myosin II inhibitor blebbistatin. After 9-h release from thymidine block at G1/S phase, addition of nocodazole at 20 ng/ml but not 40 ng/ml ensures rapid release from the nocodazole arrest. Subsequently, the cells are cultured in the presence of 50 μM blebbistatin for 20 and 50 min to enrich cells in anaphase and telophase, respectively. Western blot analysis verifies down-regulation of phospho-histone H3-Ser10, phospho-Aurora A/B/C, and cyclin B1 during M-phase progression. Furthermore, we show how the electrophoretic mobility shifts of the Src-family kinases c-Yes and c-Src can change in each phase of mitosis. These results provide a useful synchronization method for biochemically examining protein dynamics during M-phase progression.     

Localization of MPM-2 recognized phosphoproteins and tubulin during cell cycle progression in synchronized Vicia faba root meristem cells.

MPM-2 antibody reacts with a subset of mitotic phosphoproteins. We followed localization of MPM-2 immunoreactive material and localization of microtubules during cell cycle progression in a highly synchronous population of Vicia faba root meristem cells and isolated nuclei. The MPM-2 antibody labelling showed significant cell cycle dependence. MPM-2 nuclear reactivity was weak and homogeneous in G1 and S phase of the cell cycle and became stronger and heterogeneous during G2, resembling staining of the nuclear matrix, with maximum staining at the G2/M interface. Similarly the staining intensity of nucleoli increased from late G1 phase to nucleoli dispersion in early prophase. During mitosis MPM-2 immunoreactivity was associated with spindle configurations and the brightest signal was localized in kinetochores from prophase to metaphase.     

Immediate effects of serum depletion on dissociation between growth in size and cell division in proliferating 3T3 cells

Proliferating nonconfluent 3T3 cells become committed to proceed through the cell cycle or to enter G0 during the first post-mitotic part of G1 (G1pm). The decision to proceed through G1pm is dependent on the presence of serum growth factors in the culture medium. Cells that have passed this particular growth-factor-dependent cell cycle stage are independent of serum growth factors and undergo mitosis on schedule. We report here that G1ps, S, and G2 cells cease to increase in size when serum is withdrawn. As a result the mitotic cell size after 8 hours serum starvation is reduced to approximately 60% of the normal mitotic cell. This reduced growth in cell size is due to a rapid decrease in protein synthesis and some increase in protein degradation. This dissociation between growth in size and cell-cycle progression within a single cell cycle provides a new approach to study the two processes separately.     

Staurosporine引起正常和肿瘤细胞内Ca~(2 )和CaM水平发生变化

低剂量Staurosporine可以使正常细胞可逆地阻断于G1期,但对肿瘤细胞的周期运行不发生任何影响。本文利用显微光度术,测定了单个细胞内Ca~(2 )、活化钙调素和总钙调素的含量,结果表明:5ng/mL staurosporine作用于细胞18h,使正常细胞2BS G1和S期总CaM含量降低;而BGC-823细胞各周期时相总钙调素含量不发生改变;钙活化钙调素增加。Staurosporine阻断2BS细胞于G1期而不影响BGC-823的周期运行可能与Stauro-Sporine使2BS G1期细胞的钙调素降低以及抑制了2BS细胞的P~(107)磷酸化有关。     

The paramyxovirus simian virus 5 V protein slows progression of the cell cycle

Infection of cells by many viruses affects the cell division cycle of the host cell to favor viral replication. We examined the ability of the paramyxovirus simian parainfluenza virus 5 (SV5) to affect cell cycle progression, and we found that SV5 slows the rate of proliferation of HeLa T4 cells. The SV5-infected cells had a delayed transition from G(1) to S phase and prolonged progression through S phase, and some of the infected cells were arrested in G(2) or M phase. The levels of p53 and p21(CIP1) were not increased in SV5-infected cells compared to mock-infected cells, suggesting that the changes in the cell cycle occur through a p53-independent mechanism. However, the phosphorylation of the retinoblastoma protein (pRB) was delayed and prolonged in SV5-infected cells. The changes in the cell cycle were also observed in cells expressing the SV5 V protein but not in the cells expressing the SV5 P protein or the V protein lacking its unique C terminus (VDeltaC). The unique C terminus of the V protein of SV5 was shown previously to interact with DDB1, which is the 127-kDa subunit of the multifunctional damage-specific DNA-binding protein (DDB) heterodimer. The coexpression of DDB1 with V can partially restore the changes in the cell cycle caused by expression of the V protein.