Cyclin dependent kinases and their role in regulation of plant cell cycle

Plants have capability to optimize its architecture by using CDK pathways. It involves diverse types of cyclin dependent kinase enzymes (CDKs). CDKs are classified in to eight classes (CDKA to CDKG and CKL) based on the recognized cyclin-binding domains. These enzymes require specific cyclin proteins to get activated. They form complex with cyclin subunits and phosphorylate key target proteins. Phosphorylation of these target proteins is essential to drive cell cycle further from one phase to another phase. During cell division, the activity of cyclin dependent kinase is controlled by CDK interactor/inhibitor of CDKs (ICK) and Kip-related proteins (KRPs). They bind with specific CDK/cyclin complex and help in controlling CDKs activity. Since cell cycle can be progressed further only by synthesis and destruction of cyclins, they are quickly degraded using ubiquitination-proteasome pathway. Ubiquitylation reaction is followed by DNA duplication and cell division process. These two processes are regulated by two complexes known as Skp1/cullin/F-box (SCF)-related complex and the anaphase-promoting complex/cyclosome (APC/C). SCF allows cell to enter from G1 to S phase and APC/C allows cell to enter from G2 to M phase. When all these above processes of cell division are going on, genes of cyclin dependent kinases gets activated one by one simultaneously and help in regulation of CDK pathways. How cell cycle is regulated by CDKs is discussed.     

Key events in the cell cycle, their regulation and organization

The review surveys the studies of molecular genetic mechanisms of the cell cycle control on various eukaryotic models. The major cell cycle phenomena are considered: (1) checkpoints and their role in preserving DNA integrity and fidelity of mitosis, (2) the cell oscillator model, and (3) the role of cyclins in timing of cell division and coordination of mitotic events. The main classes of regulatory proteins involved in the cell cycle are discussed in detail.     

A rapid survival assay to measure drug-induced cytotoxicity and cell cycle effects

We describe a rapid method to accurately measure the cytotoxicity of mammalian cells upon exposure to various drugs. Using this assay, we obtain survival data in a fraction of the time required to perform the traditional clonogenic survival assay, considered the gold standard. The dynamic range of the assay allows sensitivity measurements on a multi-log scale allowing better resolution of comparative sensitivities. Moreover, the results obtained contain additional information on cell cycle effects of the drug treatment. Cell survival is obtained from a quantitative comparison of proliferation between drug-treated and untreated cells. During the assay, cells are treated with a drug and, following a recovery period, allowed to proliferate in the presence of bromodeoxyuridine (BrdU). Cells that synthesize DNA in the presence of BrdU exhibit quenched Hoechst fluorescence, easily detected by flow cytometry; quenching is used to determine relative proliferation in treated vs. untreated cells. Finally, this assay can be used in high-throughput format to simultaneously screen multiple cell lines and drugs for accurate measurements of cell survival and cell cycle effects after drug treatment.     

Selective effects of purine and pyrimidine analogues and of respiratory inhibitors on perithecial development and branching in sordaria

The initiation of perithecia in the homothallic ascomycete Sordaria fimicola was completely suppressed, without seriously inhibiting vegetative growth, by growing the fungus on an agar medium containing one of the following additions: 1) 1 μm 5-fluorouracil, 2) 10 to 100 μm 6-azauracil, 8-azaguanine or 8-azaadenine, 3) 50 to 500 μm cyanide or azide, 4) 5% (w/v) casein hydrolysate. In contrast to the selective activity of the analogues of 3 RNA bases, whose inhibition could be reversed by the appropriate normal bases only, none of the analogues of thymine were active, neither were the thio-derivatives of RNA bases. Other inhibitors of RNA and protein synthesis, like actinomycin D, puromycin and cycloheximide, were also without selective activity, although the last of these inhibited perithecial maturation at 0.1 μm concentration but not initiation. Amino acid analogues were inactive, as were the metabolic inhibitors thiourea, 2,4-dinitrophenol and fluoride. The compounds which inhibited the formation of perithecia also lowered the branching frequency of leading hyphae, but not their linear growth rates. Consequently, the branch densities were diminished in their presence. Hypotheses to account for these findings are discussed in terms of inhibition of growth in general, of the synthesis of some specific messenger RNAs, and of RNA-mediated transport across membranes, the last of which seeming the most fruitful for further work.     

Carbamates of 4'-demethyl-4-deoxypodophyllotoxin: synthesis, cytotoxicity and cell cycle effects

In an attempt to generate compounds with superior bioactivity and reduced toxicity, 12 carbamates of 4′-demethyl-4-deoxypodophyllotoxin, N-(1-oxyl-4′-demethyl- 4-deoxypodophyllic)-α-amino acids amides, were synthesized and evaluated for antiproliferative activity and cell cycle effects. These synthesized compounds proved to be more hydrophilic, as well as improved or comparable in vitro cytotoxicities against four cell lines (A-549, HeLa, SiHa, and HL-60) compared with either parent DPT or anti-cancer drug VP-16. Furthermore, flow cytometric analysis exhibited that N-(1-oxyl-4′-demethyl-4-deoxypodophyllic)-d-α-methine amide (15f) induced cell cycle arrest in the G2/M phase in A-549 cells.     

Monoamine oxidase inhibitors, their structural analogues, and neuroprotection

Monoamine oxidase (MAO) inhibitors have been used for many years in the treatment of psychiatric and neurological disorders. More recently, some of these drugs and their analogues have been shown to have neuroprotective and neurorescue effects in several models of neurologic insult, including in vitro and in vivo models of cerebral ischemia. This review will discuss current evidence regarding these aspects of l-deprenyl, tranylcypromine, phenelzine, and some structurally related drugs.     

The role of stathmin in the regulation of the cell cycle

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.     

Fluorination of triptolide and its analogues and their cytotoxicity

The reaction of triptolide and its analogues with a fluorinating agent, that is, bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-Fluor) or (diethylamino)sulfur trifluoride (DAST), was studied. One of the fluorinated products, 14beta-dehydroxy-14beta-fluoro triptolide, was found to be more cytotoxic than the parent natural triptolide.     

Synthesis of hybrid acetogenins, alpha,beta-unsaturated-gamma-lactone-free nitrogen-containing heterocyclic analogues, and their cytotoxicity against human cancer cell lines

A series of alpha,beta-unsaturated-gamma-lactone-free nitrogen-containing heterocyclic analogues of solamin, a natural mono-THF acetogenin, have been synthesized and their cytotoxicity was investigated against 39 tumor cell lines. One of them, 1-methylpyrazol-5-yl derivative, showed selective increase of cytotoxicity against NCI-H23 with 80 times higher potency than solamin.     

Differential and kinetic effects of cell cycle inhibitors on neoplastic and primary astrocytes

Alterations in cell cycle regulation underlie the unrestricted growth of neoplastic astrocytes. Chemotherapeutic interventions of gliomas have poor prognostic outcomes due to drug resistance and drug toxicity. Here, we examined the in vitro growth kinetics of C6 glioma (C6G) cells and primary astrocytes and their responses to 2 phase-specific inhibitors, lovastatin and hydroxyurea. C6G cells demonstrated a shorter G₁ phase and an earlier peak of DNA synthesis in S phase than primary astrocytes. As C6G cells and primary astrocytes re-entered the cell cycle in the presence of lovastatin or hydroxyurea, they exhibited different sensitivities to the inhibitory effects of these agents, as measured by [³H]-thymidine incorporation. Compared to primary astrocytes, C6G cells were more sensitive to lovastatin, but less sensitive to hydroxyurea. Studies using 2 different paradigms of exposure uncovered dramatic differences in the kinetics of DNA synthesis inhibition by these 2 agents in C6G cells and primary astrocytes. One notable difference was the ability of C6G cells to more easily recover from the inhibitory effects of hydroxyurea following short exposure. Our results provide insight into C6 glioma drug resistance as well as the inhibitory effects of these 2 phase-specific inhibitors and their chemotherapeutic potential.     

Thermal 7-9 transglycosylation of purine nucleosides and their analogues

A novel thermal 7 in equilibrium 9 transglycosylation reaction was studied in the series of fully acetylated purine ribonucleosides and their 2-acetoxyethoxymethyl analogues. Ratio 7- to 9-isomers in the resultant mixtures was determined by the proton magnetic resonance spectroscopy.     

Pyrimidine nucleosides enhance the efficacy of inhibitors of pyrimidine biosynthesis in cultured hepatocellular carcinoma cells

Inhibition of colony formation in cultured hepatocellular carcinoma cells of the rat was used to test the efficacy of inhibitors of de novo pyrimidine biosynthesis as potential anticancer drugs. N-(phosphonacetyl)-L-aspartic acid (PALA) (10 and 100 micrograms/ml) and 5-aza-5,6-dihydroorotic acid (DHOX) (100 micrograms/ml) inhibited the formation of colonies and these inhibitions were completely reversed by inclusion of 0.1 mM uridine, the end product of de novo pyrimidine biosynthesis, in the culture medium. With some lots of fetal bovine serum where PALA and DHOX had little effect on inhibiting colony formation, addition of 0.1 mM cytidine restored the inhibitory characteristics of PALA and, to some extent, DHOX. The results demonstrate that cytidine levels modulate the inhibitions of hepatoma colony formation by both PALA and DHOX and that co-administration of these drugs together with cytidine provides a simple expedient to increase drug efficacy.     

Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors.

In this study, we explored what effect inhibitors of the 26S proteasome have on cell cycle distribution and induction of apoptosis in human skin fibroblasts and colon cancer cells differing in their p53 status. We found that proteasome inhibition resulted in nuclear accumulation of p53. This was surprising because it is thought that the degradation of p53 is mediated by cytoplasmic 26S proteasomes. Nuclear accumulation of p53 was accompanied by the induction of both p21WAF1 mRNA and protein as well as a decrease in cells entering S phase. Interestingly, cells with compromised p53 function showed a marked increase in the proportion of cells in the G2-M phase of the cell cycle and an attenuated induction of apoptosis after proteasome inhibition. Taken together, our results suggest that proteasome inhibition results in nuclear accumulation of p53 and a p53-stimulated induction of both G1 arrest and apoptosis.     

Small-molecule inhibitors of the cell cycle

The cell cycle remains an attractive target for the development of small-molecule inhibitors for use as both novel chemotherapeutics and research probes. Given the importance of cytoskeletal dynamics and cyclin-dependent kinases for cell-cycle progression, much interest has focused on the identification of anti-mitotic agents and kinase inhibitors. However recent advances in cell-based screening technologies and an increased interest in inhibitors with greater specificity are beginning to influence the search for novel cell-cycle inhibitors.     

Auxin regulation of cell cycle and its role during lateral root initiation

The plant hormone auxin plays a crucial role in the upstream regulation of many processes, making the study of its action particularly interesting to understand plant development. In this review we will focus on the effects auxin exerts on cell cycle progression, more specifically, during the initiation of lateral roots. Auxin fulfils a dominant role in the initiation of a new lateral root primordium. How this occurs remains largely unknown. Here we try to integrate the classical auxin signalling mechanisms into recent findings on cell cycle regulation. How both signalling cascades are integrated appears to be complex and is far from understood. As a means to solve this problem we suggest the use of a lateral root-inducible system that allows investigation of the early signalling cascades initiated by auxin and leading to cell cycle activation.     

A model for regulation of the cell cycle incorporating cyclin A, cyclin B and their complexes

Abstract. t. A mathematical model for the cell cycle is proposed that incorporates the known biochemical reactions involving both cyclin A and cyclin B, the interactions of these cyclins with cdc2 and cdk2, and the controlling effects of cdc25 and weel. The model also postulates the existence of an as yet unknown phosphatase involved in the formation of maturation promoting factor. The model produces solutions that agree qualitatively with a wide variety of experimentally observed cell-cycle behavior. Conditions under which the model could explain the initial rapid divisions of embryonic cells and the transition to the slower somatic cell cycle are also discussed.     

A stochastic, molecular model of the fission yeast cell cycle: role of the nucleocytoplasmic ratio in cycle time regulation

We propose a stochastic version of a recently published, deterministic model of the molecular mechanism regulating the mitotic cell cycle of fission yeast, Schizosaccharomyces pombe. Stochasticity is introduced in two ways: (i) by considering the known asymmetry of cell division, which produces daughter cells of slightly different sizes; and (ii) by assuming that the nuclear volumes of the two newborn cells may also differ. In this model, the accumulation of cyclins in the nucleus is proportional to the ratio of cytoplasmic to nuclear volumes. We have simulated the cell-cycle statistics of populations of wild-type cells and of wee1(-) mutant cells. Our results are consistent with well known experimental observations.