Inhibitor R-roscovitine Research Articles

Safety and pharmacokinetics of Roscovitine (Seliciclib) in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, a randomized, placebo-controlled study

BackgroundThe orally available kinase inhibitor R-roscovitine has undergone clinical trials against various cancers and is currently under clinical evaluation against Cushing disease and rheumatoid arthritis. Roscovitine displays biological properties suggesting potential benefits in CF: it partially corrects F508del-CFTR trafficking, stimulates the bactericidal properties of CF alveolar macrophages, and displays anti-inflammatory properties and analgesic effects. MethodsA phase 2 trial study (ROSCO-CF) was launched to evaluate the safety and effects of roscovitine in Pseudomonas aeruginosa infected adult CF patients carrying two CF causing mutations (at least one F508del-CFTR mutation) and harboring a FEV1 ≥40%. ROSCO-CF was a multicenter, double-blind, placebo-controlled, dose-ranging study (200, 400, 800 mg roscovitine, orally administered daily for 4 days/week/4 weeks). ResultsAmong the 34 volunteers enrolled, randomization assigned 11/8/8/7 to receive the 0 (placebo)/ 200/400/800 mg roscovitine doses, respectively. In these subjects with polypharmacy, roscovitine was relatively safe and well-tolerated, with no significant adverse effects (AEs) other than five serious AEs (SAEs) possibly related to roscovitine. Pharmacokinetics of roscovitine were rather variable among subjects. No significant efficacy, at the levels of inflammation, infection, spirometry, sweat chloride, pain and quality of life, was detected in roscovitine-treated groups compared to the placebo-treated group. ConclusionRoscovitine was relatively safe and well-tolerated in CF patients especially at the 200 and 400 mg doses. However, there were 5 subject withdrawals due to SAEs in the roscovitine group and none in the placebo group. The lack of evidence for efficacy of roscovitine (despite encouraging cellular and animal results) may be due to high pharmacokinetics variability, short duration of treatment, and/or inappropriate dosing protocol.

Induction of Apoptosis in G1/S Blocked HeLa Cells by R-Roscovitine: A Preliminary Study

HeLa cells are human cervical cancer cells with HR HPV-18 genes integrated in the genome. The functions of tumor suppressor proteins p53 and pRB are abrogated and cell cycle regulation becomes nonfunctional. The aim of the present study was to investigate whether the CDK inhibitor R-Roscovitine would allow the G1/S blocked HeLa cells to enter into mitosis prematurely and induce apoptosis. HeLa cells blocked in G1/S border were treated with different concentrations of Roscovitine for 4 and 18 h respectively. Induction of apoptosis was studied by FACS and DNA fragmentation. Presence of γH2AX in the treated cells was studied by confocal microscopy. Expression levels of CASP3, CDKN1A i.e. p21 (Cip1/Waf1) and Bcl2 were studied by semi-quantitative RT-PCR to analyze the role played by these proteins in Roscovitine induced apoptosis in G1/S blocked HeLa cells. Results indicate that the Roscovitine allowed the thymidine blocked HeLa cells to enter into mitosis prematurely. Presence of γH2AX loci in treated cells indicates DNA damage in prematurely mitotic cells. Analysis of DNA fragmentation and chromatin condensation confirmed apoptosis as the possible mechanism of Roscovitine induced cell death. Our results also reveal that Roscovitine induced apoptosis is associated with the overexpression of CASP3, p21 (cip1/waf1) and Bcl2.

Cyclin-Dependent Kinase Inhibition Reduces Lung Damage in a Mouse Model of Ventilator-Induced Lung Injury

Mechanical ventilation (MV) has the potential to induce lung damage in healthy lungs or aggravate existing lung injury. Polymorphonuclear neutrophil (PMN) recruitment plays an important role in driving the inflammatory response in ventilator-induced lung injury (VILI). The cyclin-dependent kinase inhibitor r-roscovitine has been shown to induce apoptosis in PMNs. In this study, we investigated the potential of r-roscovitine treatment in reducing lung damage in a mouse model of VILI. Mice were tracheotomized and subjected to lung-protective MV with lower (∼7.5 mL/kg) or lung-injurious MV with higher (∼15 mL/kg) tidal volume (VT). R-roscovitine treatment enhanced apoptosis in PMNs in vitro. Ventilator-induced lung injury was associated with pulmonary PMN influx in low and high VT MV. During lung-injurious MV, r-roscovitine treatment reduced the number of PMNs and lowered levels of the lung damage markers RAGE (receptor for advanced glycation end products) and total immunoglobulin M in bronchoalveolar lavage fluid. R-roscovitine did not affect cytokine or chemokine levels in the bronchoalveolar space, neither during lung-protective nor lung-injurious MV. Thus, r-roscovitine treatment reduces lung damage in VILI, possibly dependent on increased apoptosis of PMNs.

Cyclin-Dependent Kinase 9 Activity Regulates Neutrophil Spontaneous Apoptosis

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

Can ex vivo evaluation (testing) predict the sensitivity of CLL cells to therapy with purine analogs in conjunction with an alkylating agent? A comparison of in vivo and ex vivo responses to treatment

Malfunctions in the regulation of apoptosis cause the accumulation of malignant, long-lived B CD19+/CD5+ cells in chronic lymphocytic leukemia (CLL). The primary goal in CLL therapy is to overcome resistance to apoptosis and efficiently trigger programmed cell death in leukemic cells. This study demonstrated that the in vivo responses of malignant cells from CLL patients after administration of purine analogs (cladribine/fludarabine) with cyclophosphamide vary significantly. For comparative purposes, the sensitivity of leukemic cells obtained from the same CLL patients to conventional purine analogs and the selective CDK inhibitor R-roscovitine (ROSC) was determined, with and without the addition of an alkylating agent, prior to the onset of in vivo therapy. The kinetics and rate of spontaneous and drug-induced apoptosis of CLL cells under ex vivo conditions differed significantly between patients, mirroring the variability observed during in vivo treatment. Interestingly, individual patients' leukemic cells were comparably sensitive to the drugs under both conditions. Of the drugs examined, ROSC exerted the highest therapeutic efficacy under ex vivo conditions. Our results indicate that ex vivo testing might be useful for identifying the most potent first-line therapeutic regimen for specific CLL patients and possibly for the design of therapies tailored for individual CLL patients.

Effects of the cyclin‐dependent kinase inhibitor R‐roscovitine on eosinophil survival and clearance

Eosinophils are pro-inflammatory cells implicated in the pathogenesis of asthma and atopy. Apoptosis has been proposed as a potential mechanism underlying the resolution of eosinophilic inflammation and studies have indicated the ability of interventions that induce human eosinophil apoptosis to promote the resolution of eosinophilic inflammation. Recently, the cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to enhance neutrophil apoptosis and promote the resolution of neutrophilic inflammation. The purpose of this study was to examine the expression of CDKs in human blood eosinophils, the effects of R-roscovitine on eosinophil survival in vitro and whether R-roscovitine could influence eosinophilic lung inflammation in vivo. Eosinophils were isolated from human peripheral blood and the effects of R-roscovitine on apoptosis, degranulation and phagocytic uptake examined in vitro. The effects of R-roscovitine on eosinophilic lung inflammation in vivo were also assessed using an ovalbumin mouse model. Our data demonstrate that human eosinophils express five known targets for R-roscovitine: CDK1, -2, -5, -7 and -9. R-roscovitine induced eosinophil apoptosis in a time- and concentration-dependent manner but also accelerated transition to secondary necrosis as assessed by microscopy, flow cytometry and caspase activation. In addition, we show that R-roscovitine can override the anti-apoptotic signals of GM-CSF and IL-5. We report that the pro-apoptotic effect of R-roscovitine is associated with suppression of Mcl-1L expression and that this compound enhanced phagocytic clearance of eosinophils by macrophages. Finally, we show that R-roscovitine induces apoptosis in murine peripheral blood and spleen-derived eosinophils; despite this, R-roscovitine did not modulate the tissue and lumen eosinophilia characteristic of the ovalbumin mouse model of airway eosinophilia. These data demonstrate that R-roscovitine is capable of inducing rapid apoptosis and secondary necrosis in eosinophils but does not affect the onset or improve the resolution of eosinophilic airway inflammation in vivo.

S34 Effects of the cyclin-dependent kinase inhibitor R-roscovitine on eosinophil survival and clearance

BackgroundEosinophils are pro-inflammatory cells implicated in the pathogenesis of asthma and atopy. Apoptosis has been proposed as a potential mechanism underlying the resolution of eosinophilic inflammation and studies have indicated...

Cyclin-dependent kinase inhibitor r-roscovitine reduces lipoteichoic acid lung inflammation and improves the resolution of antibiotic-treated Streptococcus pneumoniae pneumonia

Streptoccocus pneumoniae pneumonia remains associated with high morbidity and mortality. Antibiotic treatment frequently is insufficient in limiting lung damage due to inflammation. Therefore, additional treatment strategies are needed. The drug r-roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was demonstrated to reduce inflammation in several models of inflammation.

Immunological Consequences of Apoptotic Cell Phagocytosis

Cells undergo apoptosis in development, tissue homeostasis, and disease and are subsequently cleared by professional and nonprofessional phagocytes. There is now overwhelming evidence that phagocyte function is profoundly altered following apoptotic cell uptake, with consequences for the ensuing innate and adaptive immune response. Pathogens and tumors exploit the changes in macrophage function following apoptotic cell uptake. Here, we will outline the consequences of apoptotic cell phagocytosis and illustrate how apoptotic cells could be used to manipulate the immune response for therapeutic gain.

Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

Dynamic Recruitment of NF-Y and Histone Acetyltransferases on Cell-cycle Promoters

Regulation of transcription during the cell-cycle is under the control of E2 factors (E2Fs), often in cooperation with nuclear factor Y (NF-Y), a histone-like CCAAT-binding trimer. NF-Y is paradigmatic of a constitutive, ubiquitous factor that pre-sets the promoter architecture for other regulatory proteins to access it. We analyzed the recruitment of NF-Y, E2F1/4/6, histone acetyltransferases, and histone deacetylase (HDAC) 1/3/4 to several cell-cycle promoters by chromatin immunoprecipitation assays in serum-starved and restimulated NIH3T3 cells. NF-Y binding is not constitutive but timely regulated in all promoters tested, being displaced when promoters are repressed. p300 association correlates with activation, and it is never found in the absence of NF-Y, whereas PCAF/hGCN5 is often found before NF-Y association. E2F4 and E2F6, together with HDACs, are bound to repressed promoters, including the G2/M Cyclin B2. As expected, an inverse relationship between HDACs association and histones H3/H4 acetylation is observed. Blocking cells in G1 with the cyclin-dependent kinase 2 inhibitor R-roscovitine confirms that NF-Y is bound to G1/S but not to G2/M promoters in G1. These data indicate that following the release of E2Fs/HDACs, a hierarchy of PCAF-NF-Y-p300 interactions and H3-H4 acetylations are required for activation of cell-cycle promoters.