We propose an integrated computational model for the network of cyclin-dependent kinases (Cdks) that controls the dynamics of the mammalian cell cycle. The model contains four Cdk modules regulated by reversible phosphorylation, Cdk inhibitors, and protein synthesis or degradation. Growth factors (GFs) trigger the transition from a quiescent, stable steady state to self-sustained oscillations in the Cdk network. These oscillations correspond to the repetitive, transient activation of cyclin D/Cdk4 -6 in G 1, cyclin E/Cdk2 at the G 1/S transition, cyclin A/Cdk2 in S and at the S/G2 transition, and cyclin B/Cdk1 at the G2/M transition. The model accounts for the following major properties of the mammalian cell cycle: (i) repetitive cell cycling in the presence of suprathreshold amounts of GF; (ii) control of cell-cycle progression by the balance between antagonistic effects of the tumor suppressor retinoblastoma protein (pRB) and the transcription factor E2F; and (iii) existence of a restriction point in G 1, beyond which completion of the cell cycle becomes independent of GF. The model also accounts for endoreplication. Incorporating the DNA replication checkpoint mediated by kinases ATR and Chk1 slows down the dynamics of the cell cycle without altering its oscillatory nature and leads to better separation of the S and M phases. The model for the mammalian cell cycle shows how the regulatory structure of the Cdk network results in its temporal self-organization, leading to the repetitive, sequential activation of the four Cdk modules that brings about the orderly progression along cell-cycle phases. cellular rhythms ͉ oscillations ͉ mitotic oscillator ͉ model ͉ systems biology I n the presence of sufficient amounts of growth factor (GF), mammalian cells quit a quiescent state, denoted G 0 , and start their progression in the cell cycle (1-3). During the G 1 phase, cells pass the restriction point, which is a point of no return beyond which they are irreversibly engaged in the cell cycle and do not require the presence of GF to complete mitosis (1, 2). Progression in the cell cycle is controlled by the sequential, transient activation of a family of cyclin-dependent kinases (Cdks), which allow an ordered succession of the cell-cycle phases G 1 , S, G 2 , and M (4, 5), even though there appears to be a certain overlapping of the different cyclins and Cdks (6). The Cdk proteins are active only when forming a complex with their corresponding cyclin. The cyclin D/Cdk4-6, cyclin E/Cdk2, cyclin A/Cdk2, and cyclin B/Cdk1 complexes promote, respectively, progression in G 1 , the transition to DNA replication in S, progression in S and transition to G 2 , and finally the G 2 /M transition allowing entry into mitosis (3-7). Cdk regulation is achieved through a variety of mechanisms that include association with cyclins and protein inhibitors, phosphorylationdephosphorylation (8), and cyclin synthesis or degradation (9).A number of theoretical models for the cell cycle have been proposed. Initially, these models pertained t...

A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).

The pituitary hormone thyrotropin, or thyroid-stimulating hormone (TSH), is the main physiological agent that regulates the thyroid gland. The thyrotropin receptor (TSHR) was cloned by selective amplification with the polymerase chain reaction of DNA segments presenting sequence similarity with genes for G protein-coupled receptors. Out of 11 new putative receptor clones obtained from genomic DNA, one had sequence characteristics different from all the others. Although this clone did not hybridize to thyroid transcripts, screening of a dog thyroid complementary DNA (cDNA) library at moderate stringency identified a cDNA encoding a 4.9-kilobase thyroid-specific transcript. The polypeptide encoded by this thyroid-specific transcript consisted of a 398-amino acid residue amino-terminal segment, constituting a putative extracellular domain, connected to a 346-residue carboxyl-terminal domain that contained seven putative transmembrane segments. Expression of the cDNA conferred TSH responsiveness to Xenopus oocytes and Y1 cells and a TSH binding phenotype to COS cells. The TSHR and the receptor for luteinizing hormone-choriogonadotropin constitute a subfamily of G protein-coupled receptors with distinct sequence characteristics.

The cell division cycle and the circadian clock represent two major cellular rhythms. These two periodic processes are coupled in multiple ways, given that several molecular components of the cell cycle network are controlled in a circadian manner. For example, in the network of cyclin-dependent kinases (Cdks) that governs progression along the successive phases of the cell cycle, the synthesis of the kinase Wee1, which inhibits the G2/M transition, is enhanced by the complex CLOCK-BMAL1 that plays a central role in the circadian clock network. Another component of the latter network, REV-ERBα, inhibits the synthesis of the Cdk inhibitor p21. Moreover, the synthesis of the oncogene c-Myc, which promotes G1 cyclin synthesis, is repressed by CLOCK-BMAL1. Using detailed computational models for the two networks we investigate the conditions in which the mammalian cell cycle can be entrained by the circadian clock. We show that the cell cycle can be brought to oscillate at a period of 24 h or 48 h when its autonomous period prior to coupling is in an appropriate range. The model indicates that the combination of multiple modes of coupling does not necessarily facilitate entrainment of the cell cycle by the circadian clock. Entrainment can also occur as a result of circadian variations in the level of a growth factor controlling entry into G1. Outside the range of entrainment, the coupling to the circadian clock may lead to disconnected oscillations in the cell cycle and the circadian system, or to complex oscillatory dynamics of the cell cycle in the form of endoreplication, complex periodic oscillations or chaos. The model predicts that the transition from entrainment to 24 h or 48 h might occur when the strength of coupling to the circadian clock or the level of growth factor decrease below critical values.

To understand the dynamics of the cell cycle, we need to characterize the balance between cell cycle arrest and cell proliferation, which is often deregulated in cancers. We address this issue by means of a detailed computational model for the network of cyclin-dependent kinases (Cdks) driving the mammalian cell cycle. Previous analysis of the model focused on how this balance is controlled by growth factors (GFs) or the levels of activators (oncogenes) and inhibitors (tumour suppressors) of cell cycle progression. Supra-threshold changes in the level of any of these factors can trigger a switch in the dynamical behaviour of the Cdk network corresponding to a bifurcation between a stable steady state, associated with cell cycle arrest, and sustained oscillations of the various cyclin/Cdk complexes, corresponding to cell proliferation. Here, we focus on the regulation of cell proliferation by cellular environmental factors external to the Cdk network, such as the extracellular matrix (ECM), and contact inhibition, which increases with cell density. We extend the model for the Cdk network by including the phenomenological effect of both the ECM, which controls the activation of the focal adhesion kinase (FAK) that promotes cell cycle progression, and cell density, which inhibits cell proliferation via the Hippo/YAP pathway. The model shows that GFs and FAK activation are capable of triggering in a similar dynamical manner the transition to cell proliferation, while the Hippo/YAP pathway can arrest proliferation once cell density passes a critical threshold. The results account for the dependence or independence of cell proliferation on serum and/or cell anchorage to ECM. Whether the balance in the Cdk network is tilted towards cell cycle arrest or proliferation depends on the direction in which the threshold associated with the bifurcation is passed once the cell integrates the multiple, internal or external signals that promote or impede progression in the cell cycle.