Nikolai Zhelev

In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R‐roscovitine)

CDK2 inhibitors have been proposed as effective anti‐cancer therapeutics. We show here that CYC202 (R‐roscovitine) is a potent inhibitor of recombinant CDK2/cyclin E kinase activity (IC50 = 0.10 μM) with an average cytotoxic IC50 of 15.2 μM in a panel of 19 human tumour cell lines, and we also demonstrate selectivity for rapidly proliferating cells over non‐proliferating cells. A study of the cell cycle effects of CYC202 in Lovo colorectal carcinoma cells showed that the major effect was not the predicted arrest in one part of the cycle, but rather an induction of cell death from all compartments of the cell cycle. The maximum tolerated dose given intravenously to mice was in excess of 20 mg/kg. Doses up to 2,000 mg/kg were tolerated when administered orally in mice. Following repeated intraperitoneal administration (3 times daily for 5 days) of 100 mg/kg to nude mice bearing the Lovo human colorectal tumour, CYC202 induced a significant antitumour effect with a 45% reduction in tumour growth compared to controls. A second experiment using the human uterine xenograft MESSA‐DX5 treated with orally administered CYC202 (500 mg/kg 3 times daily for 4 days) also exhibited a significant reduction in the rate of growth of the tumour (62%). These data, showing enzyme and cellular potency together with antitumour activity, confirm the potential of CDK2 inhibitors such as CYC202 as anticancer drugs.

Bioluminescent imaging of Cdk2 inhibition in vivo

Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle–dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle–dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.

Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine.

The aim of this study was to use two‐dimensional electrophoresis (2‐DE) coupled with multivariate principal component analysis (PCA) to characterize the quantitative changes in the protein composition of the CEM T‐lymphoblastic leukemia cell line after treatment with bohemine (BOH), a synthetic olomoucin‐derived cyclin‐dependent kinase inhibitor (CDKI). Cell classification, reflecting protein patterns, clearly distinguished two main groups: one group consists of 9, 12 and 24 h treated BOH cells while the second is represented by the 0 and 24 h control untreated cells and the 6 h BOH‐exposed CEM lymphoblasts. Discriminant protein spots differentially expressed in the BOH‐treated CEM cells were selected for identification by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) or electrospray ionization‐tandem MS (ESI‐MS/MS). Five of the selected protein spots were unequivocally identified as α‐enolase, triosephosphate isomerase, eukaryotic initiation factor 5A, and α‐ and β‐subunits of Rho GDP‐dissociation inhibitor 1. These proteins, all significantly downregulated in CEM T‐lymphoblast leukemia in the course of BOH treatment, are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement. These results indicate that the cellular effects of olomoucine‐derived CDKIs are not dependent on their ability to inhibit CDKs and could be mediated by several factors such as a decrease in protein synthesis and/or glycolysis which in turn diminishes the ability of cancer cells to function.

Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction.

Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus.

Saponins from Tribulus terrestris L. Are Less Toxic for Normal Human Fibroblasts than for Many Cancer Lines: Influence on Apoptosis and Proliferation:

The objective of the study was to explore the influence of saponins derived from Tribulus terrestris L. (TT) on normal human skin fibroblasts and to compare it with their anticancer properties. In this study, [3H]thymidine incorporation and MTT to assess cell proliferation and viability, respectively, and immunoblotting and HPLC analysis to explore intracellular signal transduction pathways have been used. We found that TT caused a dose-dependent decrease in [3H]thymidine incorporation into the DNA of treated fibroblast compared to the untreated controls. Viability of treated cells remained within the control levels with treatment of up to 5 μg TT/ml medium. It was significantly depressed with incubation in ≥6 μg TT/ml medium with an IC50 of 12.6 μg TT/ml of cultivating media. ERK1/2 was significantly dephosphorylated at 5 mins of incubation with TT until the 48th hour, when phosphorylation slightly recovered, but was still below the control levels. In contrast, p38 and JNK phosphorylation was positively influenced, with peaks at 1 hr and 24 hrs of incubation respectively. Phosphorylation/dephosphorylation events of SAPK/MAPK clearly correlated with Mkp-1 induction. Procaspase 3 was activated after 5 mins of incubation and coincided with a rapid actin cleavage. There was a significant decrease of putrescine concentration and a concomitant increase of spermidine and spermine at 2 mins of treatment. According to our results, TT is less toxic for normal human skin fibroblasts in comparison to many cancer lines investigated in previous studies. The molecular mechanism of this cytotoxicity involves up- and downregulation of polyamines’ homeostasis, suppression of proliferation, and induction of apoptosis. Further research in this field using animal models would help to explore and interpret the potential properties of TT as an anticancer supplement.

The role of modelling in identifying drug targets for diseases of the cell cycle

The cell cycle is implicated in diseases that are the leading cause of mortality and morbidity in the developed world. Until recently, the search for drug targets has focused on relatively small parts of the regulatory network under the assumption that key events can be controlled by targeting single pathways. This is valid provided the impact of couplings to the wider scale context of the network can be ignored. The resulting depth of study has revealed many new insights; however, these have been won at the expense of breadth and a proper understanding of the consequences of links between the different parts of the network. Since it is now becoming clear that these early assumptions may not hold and successful treatments are likely to employ drugs that simultaneously target a number of different sites in the regulatory network, it is timely to redress this imbalance. However, the substantial increase in complexity presents new challenges and necessitates parallel theoretical and experimental approaches. We review the current status of theoretical models for the cell cycle in light of these new challenges. Many of the existing approaches are not sufficiently comprehensive to simultaneously incorporate the required extent of couplings. Where more appropriate levels of complexity are incorporated, the models are difficult to link directly to currently available data. Further progress requires a better integration of experiment and theory. New kinds of data are required that are quantitative, have a higher temporal resolution and that allow simultaneous quantitative comparison of the concentration of larger numbers of different proteins. More comprehensive models are required and must accommodate not only substantial uncertainties in the structure and kinetic parameters of the networks, but also high levels of ignorance. The most recent results relating network complexity to robustness of the dynamics provide clues that suggest progress is possible.

Changes in a nuclear matrix antigen during the cell cycle: interphase and mitotic cells

We studied the behaviour in interphase and mitotic human cells of a 125 kDa (pI 6.5) antigen, associated with the nuclear matrix and detected in proliferating cells. Indirect immunofluorescence with a specific monoclonal antibody reveals that during interphase in WISH and Namalwa cells, as well as phytohaemagglutinin—stimulated lymphocytes, the antigen displays a speckled distribution in the nucleoplasm of all cells. At early prophase the fluorescence intensity of the coalesced speckles increases markedly. During metaphase and anaphase the antigen gives maximal fluorescence distributed diffusely in the nucleoplasm, while chromosomes remain negative. At anaphase and cytokinesis the antigen is still cytoplasmic, but fluorescence intensity decreases. Two‐dimensional gel electrophoresis and immunoblotting reveal that the p125/6.5 antigen displays a net increase in isolated mitotic cells as compared to interphase cells. These results suggest that the p125/6.5 protein participates in late G2 phase and G2/M transition events preparing the cell for mitosis.

Discovery and development of Seliciclib. How systems biology approaches can lead to better drug performance

Seliciclib (R-Roscovitine) was identified as an inhibitor of CDKs and has undergone drug development and clinical testing as an anticancer agent. In this review, the authors describe the discovery of Seliciclib and give a brief summary of the biology of the CDKs Seliciclib inhibits. An overview of the published in vitro and in vivo work supporting the development as an anti-cancer agent, from in vitro experiments to animal model studies ending with a summary of the clinical trial results and trials underway is presented. In addition some potential non-oncology applications are explored and the potential mode of action of Seliciclib in these areas is described. Finally the authors argue that optimisation of the therapeutic effects of kinase inhibitors such as Seliciclib could be enhanced using a systems biology approach involving mathematical modelling of the molecular pathways regulating cell growth and division.

Pharmacological inhibition of ATM by KU55933 stimulates ATM transcription

Ataxia-telangiectasia mutated (ATM) kinase is a component of a signalling mechanism that determines the process of decision-making in response to DNA damage and involves the participation of multiple proteins. ATM is activated by DNA double-strand breaks (DSBs) through the Mre11–Rad50–Nbs1 (MRN) DNA repair complex, and orchestrates signalling cascades that initiate the DNA damage response. Cells lacking ATM are hypersensitive to insults, particularly genotoxic stress, induced through radiation or radiomimetic drugs. Here, we investigate the degree of ATM activation during time-dependent treatment with genotoxic agents and the effects of ATM on phospho-induction and localization of its downstream substrates. Additionally, we have demonstrated a new cell-cycle-independent mechanism of ATM gene regulation following ATM kinase inhibition with KU5593. Inhibition of ATM activity causes induction of ATM protein followed by oscillation and this mechanism is governed at the transcriptional level. Furthermore, this autoregulatory induction of ATM is also accompanied by a transient upregulation of p53, pATR and E2F1 levels. Since ATM inhibition is believed to sensitize cancer cells to genotoxic agents, this novel insight into the mechanism of ATM regulation might be useful for designing more precise strategies for modulation of ATM activity in cancer therapy.

The D153del Mutation in GNB3 Gene Causes Tissue Specific Signalling Patterns and an Abnormal Renal Morphology in Rge Chickens

Background The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the β transducin subunit in the colour visual transduction process. A naturally occurring mutation ‘D153del’ in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye. Principal Findings Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the Gβ3 protein subunit that, together with different Gγ and Gα subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected. Significance These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.