Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans.

Calcium and calmodulin have been widely implicated in the control of cell proliferation. We have created a strain of the genetically tractable filamentous fungus, Aspergillus nidulans, that is conditional for calmodulin expression. This was accomplished by replacing the unique endogenous calmodulin gene with one regulated by the inducible alcohol dehydrogenase (alcA) gene promoter by homologous recombination. This strain cannot grow when the cells are incubated in medium containing a carbon source that represses the alcA promoter. Characterization of the arrested cells shows that 83% are blocked in the G2 phase of the cell cycle. The block is due to very low levels of calmodulin and is fully reversible upon changing to medium that contains an inducer of the alcA promoter. The rate of cell proliferation in this strain is dependent upon both the intracellular calmodulin and extracellular Ca2+ concentrations. Raising the calmodulin concentration by inducing the alcA promoter not only causes the cells to enter the proliferative cycle more quickly and to grow faster, but also decreases the concentration of extracellular Ca2+ required to support growth by 10-fold, as compared with cells grown in noninducing medium. Thus both the intracellular calmodulin and extracellular Ca2+ concentrations are important and interactive factors in regulating the nuclear division cycle of Aspergillus nidulans.     

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.     

The presence of parvalbumin in a nonmuscle cell line attenuates progression through mitosis

Based on studies that have examined the effect of calcium chelators on cells, it has been proposed that this cation plays a role in regulating cell proliferation. In this study a novel approach was used to indirectly examine the role of calcium in cell cycle progression. A cDNA for the Ca2+-binding protein parvalbumin has been expressed in mouse C127 cells, using a bovine papilloma virus-based expression vector. The normal role of parvalbumin is that of a calcium buffer in vertebrate fast twitch muscle, and the C127 cells do not normally express this protein. The presence of parvalbumin had several effects on the growth of C127 cells. The most striking phenotype was an increase in cell cycle duration which analysis showed was the result of an increase the length of G1 and mitosis (predominantly at prophase). Since changes in cell cycle duration typically occur as a result of changes in G1 duration, the observed increase in the length of mitosis is most unusual. The present results indicate that the previously observed increase in the rate of cell proliferation in cells with elevated calmodulin levels is not the result of a general increase in the level of cytoplasmic calcium-binding protein, but is specific to calmodulin. In addition, the results suggest that calcium regulates progression through mitosis by both calmodulin-dependent (metaphase transition) and -independent (prophase) mechanisms.     

A single calcium flux triggers chromosome replication, segregation and septation in bacteria: a model

Abrupt changes in the concentration of intracellular calcium, through the mediation of calmodulin, is presumed to play an essential role in many molecular processes in eukaryotes including triggering cell cycle events. Although early studies failed to establish any role for calcium in the growth of bacteria, recent studies have demonstrated that bacteria have several calcium transport systems, and an intracellular concentration of free calcium identical to that of higher organisms, which appears to fluctuate during the cell cycle. Moreover, calmodulin-like proteins have been reported in bacteria, and the growth of E. coli is sensitive to calmodulin inhibitors. In this article we propose that a single flux of calcium, abruptly raising the intracellular concentration of free calcium, is responsible for the triggering in bacteria of the major cell cycle events, initiation of DNA replication, chromosome partition and cell division. We predict that major roles in this process will involve a bacterial calmodulin-like protein and a primitive cytoskeleton. The mechanism of triggering different cell cycle events by a single calcium flux is discussed.     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A. nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.     

T型钙通道α1G亚单位在细胞增殖中的功能研究

Increases of functional T-type calcium channel (T-channel) expression have been associated with cellular proliferation although evidence for this remains controversial. In the present study, we have used a variety of cellular, molecular and electrophysiological techniques to test the hypothesis that T-type channels play a causal role in the signaling pathway leading to proliferation. The results showed that stable over-expression of alpha1G T-channel subunit in HEK-293 cells conferred a significant growth advantage. Thus, cell population doubling time was reduced to 13.7 +/- 0.3 h in alpha1G transfectants, compared to control cultures (22.1 +/- 1.1 h) and flow cytometry analysis showed that this was due to a reduction in the number of alpha1G transfectants residing in the G0/G1 phases of the cell cycle compared to controls. The selective T-type calcium channel blocker, mibefradil, induced a dose-dependent inhibition of proliferation in alpha1G tansfectants. Furthermore, the Western blotting results proved that the level of protein expression of CDK2, cyclin A and cyclin E was high in alpha1G transfectants compared to control cultures. Our results demonstrate that the T-type calcium channel provides a significant growth advantage to HEK-293 cells that might occur via effects on the G1/S cell cycle mechanism.     

Yeast calmodulin: structural and functional elements essential for the cell cycle.

The budding yeast Saccharomyces cerevisiae is a suitable organism for studying calmodulin function in cell proliferation. Genetic studies in yeast demonstrate that vertebrate calmodulin can functionally replace yeast calmodulin. In addition, expression of half of the yeast calmodulin molecule is found to be sufficient for cell growth. Characterization of conditional-lethal mutants of yeast calmodulin as well as the intracellular distribution of calmodulin have suggested that at least two cell cycle steps require calmodulin function. One is nuclear division and the other is the maintenance of cell polarity. A current focus is to understand which kinds of target proteins are involved in mediating the essential functions of yeast calmodulin in these processes. Thus far, three yeast enzymes whose activity is regulated by calmodulin have been identified.     

Mutational analysis of calmodulin in Saccharomyces cerevisiae.

Calmodulin is well characterized as an intracellular Ca2+ receptor in nonproliferating tissues such as muscle and brain. Several observations indicate that calmodulin is also required for cellular growth and division. Deletion of the calmodulin gene is a lethal mutation in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Expression of calmodulin antisense RNA in mouse C127 cells causes a transient arrest at G1 and metaphase. Although these results indicate calmodulin plays a critical function during proliferation, they do not reveal the function. S. cerevisiae offers an excellent system for identifying calmodulin functions. Because calmodulin mutants can be readily constructed by gene replacement the consequences of mutations in calmodulin can be directly examined in vivo without interference from wild-type calmodulin. The available wealth of information concerning all aspects of the yeast life cycle provides a large framework for interpretation of new results. The recent dissection of cell cycle regulation is just the latest example of the important insights provided by analyzing basic cellular processes in yeast. Whether studies of calmodulin in yeast will reveal a universal function is unknown. One encouraging result is that yeast cells relying on vertebrate calmodulin as their only source of calmodulin survive and grow well, even if the amount of vertebrate calmodulin is equivalent to the normal steady state levels of yeast calmodulin. This review discusses the varied techniques we are using to identify the functions of calmodulin in yeast. As part of the analysis, we are defining the essential elements of calmodulin structure.     

The PHOA and PHOB cyclin-dependent kinases perform an essential function in Aspergillus nidulans

Unlike Pho85 of Saccharomyces cerevisiae, the highly related PHOA cyclin-dependent kinase (CDK) of Aspergillus nidulans plays no role in regulation of enzymes involved in phosphorous acquisition but instead modulates differentiation in response to environmental conditions, including limited phosphorous. Like PHO85, Aspergillus phoA is a nonessential gene. However, we find that expression of dominant-negative PHOA inhibits growth, suggesting it may have an essential but redundant function. Supporting this we have identified another cyclin-dependent kinase, PHOB, which is 77% identical to PHOA. Deletion of phoB causes no phenotype, even under phosphorous-limited growth conditions. To investigate the function of phoA/phoB, double mutants were selected from a cross of strains containing null alleles and by generating a temperature-sensitive allele of phoA in a deltaphoB background. Double-deleted ascospores were able to germinate but had a limited capacity for nuclear division, suggesting a cell cycle defect. Longer germination revealed morphological defects. The temperature-sensitive phoA allele caused both nuclear division and polarity defects at restrictive temperature, which could be complemented by expression of mammalian CDK5. Therefore, an essential function exists in A. nidulans for the Pho85-like kinase pair PHOA and PHOB, which may involve cell cycle control and morphogenesis.     

An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans.

Previous work has shown that nimA encodes a cell cycle regulated protein kinase that is required along with the p34cdc2 histone H1 kinase (MPF) for mitosis in Aspergillus nidulans. We have now identified two other gene products required for mitosis in A.nidulans. nimT encodes a protein similar to the fission yeast cdc25 tyrosine phosphatase and is required for the conversion of pre-MPF to MPF and nimE encodes a B-type cyclin which is a subunit of MPF. A new genetic interaction between nimEcyclinB and nimTcdc25 type genes is reported. Increased copy number of nimEcyclinB can suppress mutation of nimTcdc25 and also lead to increased accumulation of tyrosine phosphorylated p34cdc2 (pre-MPF). This biochemical observation suggests an explanation for the genetic complementation. If nimEcyclinB recruits p34cdc2 for tyrosine phosphorylation to form pre-MPF it follows that increased expression of nimEcyclinB would increase the level of pre-MPF. The increased level of pre-MPF generated may then allow the mutant nimTcdc25 protein to convert enough pre-MPF to MPF and thus permit some mitotic progression. We also demonstrate that correct cell cycle regulation by the p34cdc2 protein kinase pathway is essential for correct developmental progression in A.nidulans.     

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.     

Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin

We evaluated the effect of several classes of calmodulin inhibitors on the activity of calmodulin prepared from C6 astrocytoma cells and studied the activity of these drugs as inhibitors of the growth of C6 cells in tissue culture. There was a good correlation between the activity of the drugs as inhibitors of calmodulin and their activity as inhibitors of cell growth. The most potent compounds were calmidazolium and melittin as compared to the phenothiazines, trifluoperazine, chlorpromazine, chlorpromazine-sulfoxide or the diphenylbutylpiperidine, pimozide. The mechanism by which the inhibition of calmodulin leads to the death of cells could not be attributed entirely to inhibition of the calmodulin-sensitive cyclic nucleotide phosphodiesterase. Calmodulin is a heat stable, calcium-binding protein involved in numerous biological processes. Recent evidence indicates that calcium and calmodulin may be important for cellular proliferation. For example, this protein changes in concentration during the cell cycle; is involved in the disassembly of the mitotic apparatus; is increased in concentration in rapidly growing hepatomas and in transformed fibroblasts. Weiss and co-workers demonstrated that phenothiazines and structurally similar drugs are capable of binding to and inhibiting the activity of calmodulin. It has been recently observed that certain drugs that inhibit the activity of calmodulin also inhibit the growth of malignant cells in vitro and in vivo. In these studies, however, there was no direct correlation of the effect of the drugs on the calmodulin from the cell type under investigation with cytotoxicity. To learn more about the relationship between a drug's ability to inhibit calmodulin and its antiproliferative activity, we correlated the effect of drugs on the activity of calmodulin prepared from the C6 astrocytoma cell line with their effect on cellular proliferation. Since many inhibitors of calmodulin readily cross the blood-brain barrier and since no acceptable treatment for malignancies of the central nervous system exist, we chose this cell line as a model for elucidating the potential antineoplastic effects of calmodulin inhibitors.     

Consequences of activating the calcium-permeable ion channel TRPV1 in breast cancer cells with regulated TRPV1 expression

Increased expression of specific calcium channels in some cancers and the role of calcium signaling in proliferation and invasion have led to studies assessing calcium channel inhibitors as potential therapies for some cancers. The use of channel activators to promote death of cancer cells has been suggested, but the risk of activators promoting cancer cell proliferation and the importance of the degree of channel over-expression is unclear. We developed an MCF-7 breast cancer cell line with inducible TRPV1 overexpression and assessed the role of TRPV1 levels on cell death mediated by the TRPV1 activator capsaicin and the potential for submaximal activation to promote proliferation. The TRPV1 level was a determinant of cell death induced by capsaicin. A concentration response curve with varying TRPV1 expression levels identified the minimum level of TRPV1 required for capsaicin induced cell death. At no level of TRPV1 over-expression or capsaicin concentration did TRPV1 activation enhance proliferation. Cell death induced by capsaicin was necrotic and associated with up-regulation of c-Fos and RIP3. These studies suggest that activators of specific calcium channels may be an effective way to induce necrosis and that this approach may not always be associated with enhancement of cancer cell proliferation.     

Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein

ALG-2 is a EF hand calcium binding protein with sequence homologies to calmodulin. Vito et al have shown that ALG-2 expression is required for apoptosis following a number of death stimuli,1 although nothing is known about the effectors which underlie ALG-2 function. Here we have used ALG-2 as bait in a yeast two hybrid screen of a mouse brain cDNA library. We found that ALG-2 binds to itself and to a novel protein that we call ALG-2 interacting protein X, Alix. Using co-immunoprecipitation experiments, we confirmed ALG-2/ALG-2 binding and demonstrated that this interaction is calcium independent. ALG-2/Alix interaction was also validated by co-immunoprecipitation, but in this case, the binding was found to be strictly calcium dependent. Alix seems highly conserved throughout evolution since it shows significant homologies to a putative C. elegans protein (YNK-1) and to proteins of A. nidulans (PalA) and S. cerevisiae (BRO1). Alix is a potential regulator or downstream effector of ALG-2 action.     

Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.     

Interferon affects intracellular calmodulin levels

Interferon lowers calmodulin levels in two cell lines sensitive to its antiproliferative effect. Further, in synchronized cells, interferon strongly inhibits the increase in calmodulin observed when control cells enter the S phase, and concomitantly inhibits DNA synthesis. Calmodulin has been implicated in the control of cell proliferation and an increase in this protein seems to be necessary for the progression of cells into the S phase of the cell cycle. Therefore, the effect of interferon on calmodulin content might constitute part of the molecular mechanism by which interferon inhibits DNA synthesis.     

Interactions between the developmental program and cell cycle regulation of Aspergillus nidulans

Aspergillus nidulans is a multicellular fungus being used to study developmental regulation and cell cycle regulation. Genetic and molecular mechanisms underlying both processes have been characterized. Two types of observations suggest that there is significant interaction between cell cycle and developmental regulatory mechanisms. First, A. nidulans development involves the formation of specialized cell types that contain different, but specific, numbers of nuclei that are differentially regulated for cell cycle progression. Second, mutations directly affecting nuclear division can have major affects on cell differentiation during development. In this essay we describe these interactions and point out potential mechanisms for the cross talk between morphogenesis and the cell cycle that are tractable for future experimental investigation.