Activation of human cyclin-dependent kinases in vitro.

We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin B1 were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or B1. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and B1, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin B1 was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cyclin B1 also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin B1 also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.     

Regulation of meiosis during mammalian spermatogenesis: the A-type cyclins and their associated cyclin-dependent kinases are differentially expressed in the germ-cell lineage

To begin to examine the function of the A-type cyclins during meiosis in the male, we have examined the developmental and cellular distribution of the cyclin A1 and cyclin A2 proteins, as well as their candidate cyclin-dependent kinase partners, Cdk1 and Cdk2, in the spermatogenic lineage. Immunohistochemical localization revealed that cyclin A1 is present only in male germ cells just prior to or during the first, but not the second, meiotic division. By contrast, cyclin A2 was expressed in spermatogonia and was most abundant in preleptotene spermatocytes, cells which will enter the meiotic pathway. Immunohistochemical detection of Cdk1 was most apparent in early pachytene spermatocytes, while staining intensity diminished in diplotene and meiotically dividing spermatocytes, the cells in which cyclin A1 expression was strongest. Cdk2 was highly expressed in all spermatocytes. Notably, in cells undergoing the meiotic reduction divisions, Cdk2 appeared to localize specifically to the chromatin. This was not the case for spermatogonia undergoing mitotic divisions. In the testis, cyclin A1 has been shown to bind both Cdk1 and Cdk2 but we show here that cyclin A2 binds only Cdk2. These results indicate that the A-type cyclins and their associated kinases have different functions in the initiation and passage of male germ cells through meiosis.     

Phosphorylation of human DNMT1: implication of cyclin-dependent kinases

DNA methylation plays a central role in the epigenetic regulation of gene expression during development and progression of cancer diseases. The inheritance of specific DNA methylation patterns are acquired in the early embryo and are specifically maintained after cellular replication via the DNA methyltransferase 1 (DNMT1). Recent studies have suggested that the enzymatic activity of DNMT1 is possibly modulated by phosphorylation of serine/threonine residues located in the N-terminal domain of the enzyme. In the present work, we report that cyclin-dependent kinases (CDKs) 1, 2 and 5 can phosphorylate Ser154 of human DNMT1 in vitro. Further evidence of phosphorylation of endogenous DNMT1 at position 154 by CDKs is also found in 293 cells treated with roscovitine, a specific inhibitor of CDK1, 2 and 5. To determine the importance of Ser154 phosphorylation, a mutant of DNMT1 encoding a single-point mutation at position 154 (S154A) was generated. This mutation induced a severe loss of enzymatic activity when compared to wild type DNMT1. Moreover, after treatment with 5-Aza-2′-Deoxycytidine (5-aza-dC), a faster decline in DNMT1 protein level was observed for HEK-293 cells expressing DNMT1(S154A) as compared to cells expressing wild type DNMT1. Our data suggest that phosphorylation of DNMT1 at Ser154 by CDKs is important for enzymatic activity and protein stability of DNMT1. Considering that tumour-associated cell cycle defects are often mediated by alterations in CDK activity, our results suggest that dysregulation of cell cycle via CDKs could induce abnormal phosphorylation of DNMT1 and lead to DNA hypermethylation often observed in cancer cells.     

Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.

Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins.     

Liver X receptors inhibit macrophage proliferation through downregulation of cyclins D1 and B1 and cyclin-dependent kinases 2 and 4

Macrophages serve essential functions as regulators of immunity and homeostasis, and their proliferation contributes to pathogenesis of certain disorders. In this report, we show that induction of macrophage proliferation by the growth factor M-CSF is negatively modulated by agonists that activate the nuclear receptor liver X receptor (LXR), both in vitro and in vivo. Both isoforms LXR α and β are involved in the antiproliferative actions of LXR ligands in macrophages. In contrast, M-CSF does not exert negative effects on LXR-mediated gene expression. Treatment with LXR agonists results in the accumulation of macrophages in the G(0)/G(1) phase of the cell cycle without affecting ERK-1/2 phosphorylation. The use of small interfering RNA or genetically modified mice revealed that, in contrast to other cellular models, functional expression of either the cyclin-dependent kinase inhibitor p27KIP1 or the cholesterol transporters ATP-binding cassette A1 or ATP-binding cassette G1 was not required for the antiproliferative effects of LXR agonists in macrophages. Western blot analysis revealed that protein expression of key molecules that regulate progression through the cell cycle, such as cyclins D1 and B1 and cyclin-dependent kinases 2 and 4, was downregulated upon LXR activation. These observations suggest a role for LXR agonists in limiting macrophage proliferative responses associated to pathogenic disorders.     

Chemical inhibitors of cyclin-dependent kinases

Transient activation o f cyclin-dependent kinases (CDKs) is responsible for transition through the successive phases of the cell-division cycle. Major changes in the expression and regulation of CDKs have been described in human tumours. Enzymatic screening is starting to uncover chemical inhibitors o f CDKs that arrest the cell cycle at various steps. This review summarizes our knowledge of the first generation inhibitors, their molecular mechanisms of action and their effects on the cell cycle and apoptosis, and discusses their potential as synchronizing agents, as ligands for affinity chromatography and as therapeutic agents.     

Cyclins,cyclin-dependent kinases and differentiation

Cyclin-dependent kinases and their regulatory subunits, the cyclins, are known to regulate progression through the cell cycle. Yet these same proteins are often expressed in non-cycling, differentiated cells. This review surveys the available information about cyclins and cyclin-dependent kinases in differentiated cells and explores the possibility that these proteins may have important functions that are independent of cell cycle regulation.     

Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6

Cdk4 and Cdk6 are thought to be essential for initiation of the cell cycle in response to mitogenic stimuli. Previous studies have shown that Cdk4 is dispensable for proliferation in most cell types, an observation attributed to a putative compensatory role by Cdk6. Cdk6-null mice are viable and develop normally although hematopoiesis is slightly impaired. Embryos defective for Cdk4 and Cdk6 die during the late stages of embryonic development due to severe anemia. However, these embryos display normal organogenesis and most cell types proliferate normally. In vitro, embryonic fibroblasts lacking Cdk4 and Cdk6 proliferate and become immortal upon serial passage. Moreover, quiescent Cdk4/Cdk6-null cells respond to serum stimulation and enter S phase with normal kinetics although with lower efficiency. These results indicate that D-type cyclin-dependent kinases are not essential for cell cycle entry and suggest the existence of alternative mechanisms to initiate cell proliferation upon mitogenic stimulation.     

Regulation of cyclin-dependent kinases in Arabidopsis thaliana

In plants, different families of cyclin-dependent kinases (CDKs) and cyclins have been identified, indicating that also in plants the progression through the cell cycle is regulated by CDKs. In all eukaryotes, CDKs exert their activity through well-controlled phosphorylations of specific substrates on serine/threonine residues. Such post-translational modifications are universal mechanisms in signal transduction pathways. They allow the organism to differentiate, regulate growth and/or adapt to environmental changes, the latter being crucial for plants because of their sedentary life-style. This adaptation might explain the occurrence of a special CDK type with plant-specific features. This review focuses on the involvement of plant CDKs in different phases of the cell cycle in Arabidopsis thaliana and outlines their regulation by binding to other proteins, and by phosphorylation and dephosphorylation.     

Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells

Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.     

2-Aminoquinazoline inhibitors of cyclin-dependent kinases

The inhibition of cyclin-dependent kinase 4 (Cdk4) causes cell cycle arrest and restores a checkpoint that is absent in the majority of tumor cells. Compounds that inhibit Cdk4 selectively are targeted for treating cancer. Appropriate substitution of 2-aminoquinazolines is demonstrated to produce high levels of selectivity for Cdk4 versus closely related serine-threonine kinases.     

Evidence of phosphatidylinositol and diacylglycerol kinases in suspension cultured plant cells

Suspension cultured cells of Catharanthus roseus and Nicotiana tabacum, after two cycles of freezing and thawing, incorporated labeled phosphate from exogenous [γ-³²P]ATP into their phospholipid fraction. Quantitative thin layer chromatography (TLC) revealed strongly labeled phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP) and phosphatidic acid (PA), and less incorporation into phosphatidylinositol diphosphate (PIP₂). Neomycin and spermine affected the amount of phosphorylation into the different components in a similar way to that described for animal cells.     

Beta-carbolines as specific inhibitors of cyclin-dependent kinases

Harmine (3), 7-fluoro-1-methyl beta-carboline (35) and 1-(5-methyl-imidazol-4-yl) beta-carboline (41) were potent and specific inhibitors of cyclin-dependent kinases. The degree of aromaticity of the tricyclic ring and the positioning of substituents are important for inhibitory activity. While most beta-carbolines inhibited CDK2 and CDK5 to the same extent, selective inhibition against CDK2 was observed in 1-(2-chlorophenyl)- (12), 1-(2-fluorophenyl)- (15), and 1-(2-chloro-5-nitrophenyl)- (28) beta-carbolines.     

Genetic analysis of mammalian cyclin-dependent kinases and their inhibitors

Entry into the cell cycle, in particular the G1/S transition, is a tightly regulated process that involves a combination of mitogenic signaling pathways and cell cycle checkpoints. Some of the key regulators of this process are frequently altered in human cancer. Although the proteins that control the G1/S transition have been extensively studied at the biochemical level, little is known regarding their physiological role in vivo. During the last few years, a series of mouse strains carrying gene targeted mutations in key regulators of the G1/S transition have been generated. They include the Rb family of proteins and some of their downstream and upstream regulators. The latter include the regulatory (cyclin) and catalytic (Cdk) subunits of some of the kinases responsible for Rb inactivation as well as all the members of two families of cell cycle inhibitors, the INK4 and the Cip/Kip proteins. In this review, we summarize the most relevant information derived from the characterization of these strains of mice and attempt to integrate it within a functional framework of cell cycle regulation in vivo.     

Tyrosinase activities of cell-free extracts and living cells of cultured melanoma cells

Melanogenesis in the course of monolayer culture of a stably melanotic clonal line C₂M, derived from a mouse melanoma B 16, was investigated. Tyrosinase activity per cell of cell-free extracts was highest when the extract was prepared from cells in the mid-exponential phase of growth, when it was more than 6 times the activity of that prepared from a fully grown culture or a culture in the very early phase. On the other hand, the enzyme activity per cell of living cells in culture was highest in the early phase of culture and decreased rapidly to a level of less than one tenth of the maximum activity, in the stationary phase.The upper limit of population density of cultured melanoma cells permissive for melanin synthesis (2 to 3 × 10⁵ cells/cm²) was much higher than that of normal (nonneoplastic) melanocytes, which had been reported to produce melanin only under conditions of clonal growth.The relative efficiency of tyrosinase activity in situ, expressed by the ratio of tyrosinase activity in culture to that of cell-free extract, decreased rapidly in the exponential phase of growth. This decrease correlates to the cell density in the culture, and little if at all to the division rate, and suggests a suppressing mechanism of melanin synthesis working at the enzyme level.     

Characterization of novel inhibitors of cyclin-dependent kinases.

In epithelial cells progression through the G1 phase of the cell cycle and preparing the cell for the S phase is regulated by cyclin D1-cdk4. Cells that express the retinoblastoma protein (pRb) are dependent on cyclin D1-cdk4 activity for their proliferation while cells that do not express pRb are not. Overexpression of cyclin D1 and/or cdk4, and loss of expression of p16 (the natural inhibitor of cyclin D1-cdk4 activity), have been implicated in several cancers. These data suggest that the aberrant activity of cyclin D1-cdk4 correlates with the tumor phenotype. Hence, blocking cyclin D1-cdk4 activity may prove to be an effective anticancer therapy for pRb(+) tumors. In this paper, we report the identification of four novel compounds that selectively inhibit cyclin D1-cdk4 activity to various degrees. We further demonstrate that two of these compounds also selectively inhibit the target, pRb(+) tumor cells. The implications of these discoveries and their utility as anticancer agents are discussed.