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.
Citation: Wang K, Hampson P, Hazeldine J, Krystof V, Strnad M, Pechan P, et al. (2012) Cyclin-Dependent Kinase 9 Activity Regulates Neutrophil Spontaneous Apoptosis. PLoS ONE 7(1): e30128. https://doi.org/10.1371/journal.pone.0030128
Editor: François Leulier, French National Centre for Scientific Research - Université Aix-Marseille, France
Received: August 5, 2011; Accepted: December 14, 2011; Published: January 19, 2012
Copyright: © 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding from the European commission (LSHB-CT-2004-503467), the Czech Ministry of Education (MSM6198959216) and the Czech Grant Agency (301/08/1649 and 204/08/0511). JH is supported by a PhD studentship from the Biotechnology and Biological Sciences Research Council UK, and JML is a holder of an Arthritis Research UK programme grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have read the journal's policy and have the following conflicts: Paul Pechan is a director of C3Bio, which holds the patent for the use of LGR1406 as an anti-inflammatory agent (PCT/EP03/03207 filed in 2003). This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Neutrophils are the shortest-lived and most abundant leukocytes, dying by apoptosis within 5.4 days of leaving the bone marrow . They form part of the immune system's first line of defence against rapidly dividing bacteria and their functional lifespan can be extended at sites of infection via the anti-apoptotic actions of pro-inflammatory cytokines, such as GM-CSF . This process is tightly regulated in order to avoid inappropriate survival of neutrophils which can lead to chronic inflammatory diseases such as Rheumatoid arthritis . Despite the key role these cells play in innate immunity and chronic inflammatory disease, our understanding of the processes that regulate their lifespan remains incomplete. It has been established that levels of the anti-apoptotic protein Mcl-1 decline as neutrophils age and enter apoptosis  and factors that extend neutrophil lifespan, such as GM-CSF, act by increasing expression of Mcl-1 . Determining the primary cause of loss of key neutrophil Bcl-2 family proteins such as Mcl-1 is thus central to understanding the short lifespan of neutrophils.
Rossi et al reported the surprising observation that the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine increased the apoptosis of neutrophils , which are non-proliferating cells. R-roscovitine treatment also accelerated the loss of Mcl-1. The cellular target of roscovitine was suggested to be the cell cycle related cyclin-dependent kinases CDK1 or CDK2 . However, expression of cell cycle related CDKs is lost as myeloblasts differentiate towards mature neutrophils , suggesting that these CDKs are unlikely to mediate the pro-apoptotic effects of roscovitine. Crucially, this publication did not consider the involvement of the cell cycle independent CDKs and to our knowledge CDK1/2 have not been implicated in processes other than cell cycle regulation. More recently the same group investigated possible non-CDK targets of R-roscovitine, but excluded a role for off-target inhibition of MAP kinase or NF-κB signalling .
We therefore reconsidered the role of CDKs in regulating neutrophil apoptosis and Mcl-1 expression and our findings suggest that a cell cycle independent CDK, CDK9, is in fact a key regulator of neutrophil apoptosis and lifespan.
We first determined the expression of CDKs in human neutrophils and found that only three were readily detected by western blotting (Fig. 1), namely the cell cycle-independent CDKs: CDK5, CDK7 and CDK9. Of these, CDK 7 and CDK9 were the predominant CDKs present, with CDK5 present only at a very low level. The promyelocytic cell line HL60 was used as a positive control for CDK expression. We could not detect any of the cell cycle-dependent CDKs (CDK1, CDK2, CDK4 or CDK6) in neutrophils, as would be expected of non-cycling cells, though all were expressed in the proliferating promyeloid HL60 cells. This is in broad agreement with previous reports showing that promyeloid progenitor cells lose expression of cell cycle dependent CDKs as they mature and differentiate towards neutrophils .
Isolated human neutrophils (N) and promyelocytic HL60 cells (H) were assessed for expression of CDK proteins by western blotting (upper panel). Blots were scanned to determine the CDK:β-actin ratio by densitometry (lower panel; black bars = HL60, grey bars = neutrophils). Data are mean ± s.d. for three separate cell extracts.
We then attempted to confirm the published effects of the broad range CDK inhibitor R-roscovitine , , showing that this compound increased neutrophil spontaneous apoptosis in a concentration dependent manner (Fig. 2a). We also assessed apoptosis using Annexin V binding and propidium iodide staining and confirmed that cells were not induced to enter necrosis during this time frame (data not shown). The short lifespan of neutrophils in culture (1–2 days) means that they are not amenable to knockdown strategies such as transfection with siRNA. In order to determine which of the three CDKs expressed by neutrophils were involved in the regulation of apoptosis we used pharmacological CDK inhibitors with appropriate selectivity profiles. A selective inhibitor of CDK1 and CDK2, NU6102 (IC50 9 nM and 6 nM, respectively) , which does not inhibit transcriptional CDKs, (CDK7 and CDK9) in whole cells , had no effect on neutrophil apoptosis (Fig. 2a). NU6102 also has some activity towards CDK5, with an IC50 of 0.48 µM (Lan-Zhen Wang, Newcastle University, personal communication), suggesting that the transcriptional CDKs are mediating the effects of roscovitine on neutrophil apoptosis. In contrast, a novel roscovitine analogue LGR 1406 which does not inhibit CDK7  was able to increase neutrophil apoptosis (Fig. 2a). To further investigate the possible role of CDK9 we examined the effects of flavopiridol, a potent inhibitor of CDK9 , on neutrophil apoptosis. Flavopiridol was the most effective at accelerating entry of neutrophils in to apoptosis (Fig. 2b) and flavopiridol treated neutrophils showed the collapsed nuclear morphology characteristic of apoptotic neutrophils (Fig. 2c).
(a) Isolated human neutrophils were incubated with NU6102 (filled triangle), R-roscovitine (filled square), or LGR1406 (filled diamond) at the concentrations shown. Apoptosis was determined after 15h by assessment of DiOC6 uptake and flow cytometry, with DiOC6 low cells taken as apoptotic. Data are mean ± s.d. for four separate experiments. * indicates p<0.05 for treatment versus control. (b) Neutrophils were incubated with flavopiridol for 15 h at the concentrations shown and apoptosis determined by assessment of DiOC6 uptake. Data are mean ± s.d. for three separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (c) Images of Giemsa stained neutrophils cultured in the absence (control) or presence of 100 nM flavopiridol for 15 h.
Upon association with its activating partner cyclin T1, CDK9 forms P-TEFb, a general transcription factor responsible for phosphorylating the C-terminal domain (CTD) of RNA polymerase II . If CDK9 activity determines neutrophil lifespan we hypothesised that its activity and/or expression should decline as neutrophils aged prior to their entry into apoptosis. We therefore determined CDK9 expression and activity in freshly isolated neutrophils and after 9h in culture, at which time point less than 10% of neutrophils will have entered apoptosis. Figure 3a shows that expression of CDK9 was maintained as neutrophils aged, but CDK9 activity decreased dramatically prior to their entry into apoptosis (Fig. 3b). We further investigated the loss of CDK9 activity by determining expression of cyclin T1 and found that its expression was dramatically reduced by 9h in culture (Fig. 3c and 3d).
Isolated human neutrophils were cultured for 9h and (a) CDK9 protein expression and (b) enzymatic activity were determined and compared with freshly isolated cells (0h). For CDK9 activity protein was immunoprecipitated from neutrophils with a monoclonal anti-CDK9 antibody and activity determined by incorporation of [γ 32P]-ATP into the CTD of RNA polymerase II. The phosphorylated peptide was isolated on an SDS-PAGE gel and excised for scintillation counting. Data are expressed as % of the value for control freshly isolated cells and are mean ± s.d. for three separate experiments. * indicates p<0.05. (c). Isolated neutrophils were cultured for 0 h or 9 h and expression of cyclin T1 determined by western blotting. Blots were probed with an antibody to β-actin to confirm equal protein loading. The blot shown is representative of 4 separate experiments performed. (d) Western blots of CDK9 expression relative to β-actin were quantitated using densitometry.
As CDK9 is a regulator of transcription inhibition of CDK9 activity will have the most significant effect on the expression of proteins with a short half life. In the case of neutrophils this would include the key anti-apoptotic protein Mcl-1. Figure 4 shows that up to 6h post isolation the protein level of Mcl-1 protein remained stable, but by 9h the level of Mcl-1 began to decline and was only 20% of the control level by 20h in culture. Inhibition of CDK9 with flavopiridol accelerated the loss of Mcl-1 protein (Fig. 4a), confirming reports of the effects of flavopiridol on Mcl-1 mRNA levels , . Furthermore, loss of Mcl-1 protein correlated with the increased neutrophil apoptosis seen over the same time scale (Fig. 4b). Measurement of CDK9 enzymatic activity confirmed that flavopiridol had inhibited the enzyme in neutrophils (Fig. 4c). To determine if other Bcl-2 family proteins expressed in neutrophils were also affected by CDK9 inhibition we determined expression of Bcl2A during in vitro culture and found that its expression did not change during a 20h period. (Fig. 4d). In addition inhibition of CDK9 with flavopiridol did not result in loss of Bcl2A (Figure 4d).
(a) Isolated human neutrophils were cultured in the absence (control) or presence of 100 nM flavopiridol for 0-20 h and Mcl-1 protein expression measured by western blotting. Blots were probed for β-actin to confirm equal loading. (b) In the same experiments neutrophil apoptosis was determined by measurement of DiOC6 uptake. Data are expressed as the % of apoptotic cells in the control (solid line) and flavopiridol (broken line) treated cultures and are mean ± s.d. for four separate experiments. * indicates p<0.05 and ** indicates p<0.01 for control versus flavopiridol treated cells. (c) CDK9 activity was measured in control, flavopiridol (100 nM) and flavopiridol plus GM-CSF (50 ng/ml) treated neutrophils by immunoprecipitating CDK9 from neutrophils with a monoclonal anti-CDK9 antibody and determining activity by assessing incorporation of [γ 32P]-ATP into the CTD of RNA polymerase II. The phosphorylated peptide was isolated on an SDS-PAGE gel and visualised by autoradiography. The image shown is representative of 3 separate experiments performed. (d) Isolated human neutrophils were cultured in the absence (control) or presence of 100 nM flavopiridol for 0–20 h and Bcl2A protein expression measured by western blotting. Blots were probed for β-actin to confirm equal loading.
Neutrophils receive survival signals at sites of inflammation from cytokines such as GM-CSF and type 1 interferons. These cytokines mediate their effects via upregulation of survival genes such as Mcl-1 and Bcl-XL , . We therefore assessed the effects of flavopiridol on neutrophil apoptosis in the presence of GM-CSF. Figure 5a shows that flavopiridol induced neutrophil apoptosis even in the presence of GM-CSF and was able to prevent the upregulation of Mcl-1 protein by GM-CSF (Fig. 5b). Unsurprisingly then GM-CSF was also not able to overcome the inhibition of CDK9 activity by flavopiridol (Figure 4c).
(a). Neutrophils were incubated with medium alone, or medium containing 100 nM flavopiridol in the absence (open bars) or presence of (filled bars) of 50 ng/ml GM-CSF. Live cells were detected by their ability to retain DiOC6 and data are mean ± s.d. of 3 separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (b). The same cells were extracted and measured for Mcl-1 content by western blotting. Blots were probed for β-actin to confirm equal loading.
Taken together these results identify CDK9 as a key regulator of the expression of Mcl-1, and thus of neutrophil apoptosis and lifespan.
Neutrophils form the first line of defence against pathogens such as rapidly dividing bacteria and fungi. They are produced by the bone marrow in vast numbers, approximately 1011 are released into the circulation each day and this can more than double at times of infection. This significant endeavour is due in great part to the very short lifespan of neutrophils, which die by apoptosis within approximately 5 days of their production . Although the short lifespan of neutrophils has been known for over 30 years, the initiating event leading to the entry of neutrophils into apoptosis has not been defined. The work of the Edwards group and others , ,  has shown that the bcl-2 family protein Mcl-1 is lost as neutrophils enter apoptosis and that factors that delay neutrophil apoptosis act via increasing or maintaining expression of Mcl-1, though other Bcl-2 family proteins also respond to survival factors including Bcl-XL . However the reason for the decline in Mcl-1 itself has not been known.
Here we show that human neutrophils expressed only the cell cycle independent CDKs CDK 5, 7 and 9. These data are in broad agreement with previous reports showing that promyeloid progenitor cells lose expression of cell cycle dependent CDKs as they mature and differentiate towards neutrophils  and that neutrophils express cell cycle independent CDKs such as CDK5 . The manuscript by Rossi et al  suggested that the pro-apoptotic effects of roscovitine were due to inhibition of CDK1 and CDK2, but did not consider the cell cycle independent CDKs. We could not detect CDK1 or CDK2 in our neutrophil preparations and it may be that differences in the purity of the preparations could explain this discordance as ours were >98% pure. What our data show is not only that the transcriptional cyclin-dependent kinase CDK9 is expressed in neutrophils but also that its activity declines as they age and enter apoptosis. Moreover, inhibition of CDK9 accelerated loss of Mcl-1 and induced rapid entry into apoptosis, but did not affect expression of Bcl-2A whose protein level did not decline as cells aged and entered apoptosis. We therefore propose that CDK9 activity ultimately determines the level of Mcl-1 and its loss is the cause of the very short lifespan of neutrophils.
Rather like peeling the layers from an onion, the next question is of course why CDK9 activity declines. Neutrophils are terminally differentiated cells, which have a characteristic multi-lobed nucleus, the result of the collapse of much of their chromatin. These cells thus have a limited capability for transcription and de novo protein synthesis and it was possible that CDK9 protein levels could not be maintained as neutrophils aged. The relative stability of CDK9 protein is in agreement with previous data showing that its expression was constant in HL60 cells through the cell cycle and also did not decline as these cells differentiated towards the monocyte lineage . Further study revealed that the CDK9 co-factor cyclin T1 was lost as neutrophils aged in culture and this is therefore the likely cause of the loss of CDK9 activity . Moreover, as cyclin T1 monomeric CDK9 has been proposed to be unstable and targeted for degradation  it is possible that CDK9 protein levels will eventually also fall once cyclin T1 is lost.
The importance of these data extends beyond neutrophil biology. Inhibition of neutrophil apoptosis is associated with chronic inflammatory disease such as Adult Respiratory Distress Syndrome  and Rheumatoid Arthritis  and the bactericidal functions of these cells are thought contribute significantly to tissue damage in these conditions. Inhibition of CDK9 resulting in the removal of neutrophils from sites of inflammation could represent a rational and novel therapeutic approach to the treatment of chronic inflammatory disease. Flavopiridol (Alvocidib) in clinical trial as a treatment for cancers including pancreatic cancer  multiple myeloma  and recently Sekine et al  showed that flavopiridol was effective at reducing joint inflammation in the collagen-induced arthritis model, but did not discover the cellular target. These authors assumed that the effect was likely to be mediated by inhibition of lymphocyte proliferation or function and when this was shown not to be the cases they did not look at other cells. Our data suggest that neutrophils were the likely cellular target of flavopiridol in their model and moreover that Alvocidib should be considered as a therapy for chronic inflammatory diseases.
Materials and Methods
The study was approved by the Coventry and Warwickshire Research Ethics Committee (06/Q0201/2) and all subjects gave their written informed consent before taking part in the study.
Isolation and culture of human neutrophils
Neutrophils were isolated from the peripheral blood of healthy volunteers as previously described . Briefly, blood was collected into tubes containing EDTA and erythrocytes sedimented with 2% dextran T-500 in normal saline. Neutrophils were isolated using a discontinuous density gradient of Percoll (Sigma-Aldrich, Poole, UK). After hypotonic lysis to eliminate any remaining erythrocytes, neutrophils were resuspended in a round-bottom polypropylene tube in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Sera Laboratories International, Bolney, UK) and containing 2 mM glutamine, 100 U/ml penicillin and 100ug/ml streptomycin (Sigma-Aldrich), termed complete medium. The neutrophils were >98% pure as determined by Giemsa differential staining and light microscopy and the only contaminating cells were eosinophils.
Neutrophils were cultured for up to 20h in the presence of a range of concentrations of the CDK inhibitors R-roscovitine or flavopiridol (Sigma Aldrich, Poole, UK). A novel roscovitine analogue LGR1406  was a gift from Libor Havlíček (Institute of Experimental Botany ASCR, Prague) and NU6102 was kindly provided by D.R.Newell (University of Newcastle, UK).
Measurement of Neutrophil Apoptosis
Neutrophil apoptosis was determined by observation of cell morphology by light microscopy following Giemsa staining  and assessment of loss of mitochondrial membrane integrity using uptake of DiOC6 (Invitrogen Ltd, Paisley, UK.) measured by flow cytometric analysis . DiOC6 low cells were taken as apoptotic.
Measurement of CDK9 activity
CDK9 activity in neutrophils was determined by immunoprecipitation of CDK9 from cells followed by an in vitro kinase assay. Briefly, neutrophils were resuspended in complete RPMI 1640 medium and incubated for 0 or 9 hours at 37°C, 5%CO2. Post incubation neutrophils were lysed in 1 ml lysis buffer (50 mM Tris, 150 mM NaCl, 4 mM AEBSF, 10 ug/ml leupeptin, 10 ug/ml pepstatin, 10 ug/ml aprotonin, 1% (v/v) Triton x-100, 2 mM NAF, 1 mM DTT, 1 mM EDTA, 1 uM Cathepsin inhibitor, 1 uM calpain inhibitor, 10% Glycerol, all purchased from Sigma-Aldrich) and left on ice for 20 minutes. Lysates were homogenised using a tight fitting dounce homogeniser followed by centrifugation at 10,000 xg for 10 minutes at 4°C. Supernatants were incubated with 2.5 ug/ml of anti-CDK9 antibody (Cell Signaling Technology, New England Biobs, UK) and 100 ul of protein G beads (Miltenyi Biotec, Bisley, UK) followed by incubation on ice for 30 minutes. Following incubation the CDK9 immunoprecipitate-bead complex was combined with 25 ul of kinase assay mix (50 mM Tris, 10 mM MgCl2, 1 mM DTT, protease inhibitor cocktail (Sigma Aldrich), phosphatase inhibitors I and II (Sigma Aldrich), 1 ug RNA polymerase II CTD repeat (AbCam, Cambridge MA, USA), 1 mM ATP and 32P-γATP (2.5 µCi, GE Healthcare Ltd, Amersham, UK)) and incubated at 30°C for 1 hour. The assay mixture was then applied to the magnetic bead isolation column (Miltenyi µ column, Miltenyi Biotec) and the eluate containing the labelled substrate was collected and transferred to nitrocellulose paper which was left to air dry. The nitrocellulose was washed 3 times using 1% (v/v) phosphoric acid and radioactivity (CPM) was then measured using a liquid scintillation counter.
Measurement of CDK, cyclin T1, Bcl2A and Mcl-1 expression
Neutrophil total proteins were precipitated with 10% TCA, precipitates were washed in ice-cold acetone and resuspended in SDS-PAGE sample buffer before analysis for CDK, cyclin T1, Bcl2A and Mcl-1 content by Western blotting. Blots were probed with monoclonal antibodies to CDK1 and CDK2 (BD Transduction laboratories, Oxford, UK), CDK4, CDK5, CDK6, and CDK7 (Cell Signaling Technology), or CDK9, cyclin T1, Bcl2A and Mcl-1 (Santa Cruz Biotechnology) and isotype specific HRP-conjugated anti-mouse secondary antibodies (Dako cytomation Ltd, Ely, UK). Blots were visualised using enhanced chemiluminescence (GE Healthcare Ltd) and were probed with antibody to β-actin (Sigma-Aldrich) to confirm equal protein loading.
Conceived and designed the experiments: JML PP MS. Performed the experiments: PH KW JH. Analyzed the data: PH KW JML. Contributed reagents/materials/analysis tools: VK MS. Wrote the paper: JML.
- 1. Pillay J, den Braber I, Vrisekoop N, Kwast LM, de Boer RJ, et al. (2010) In vivo labeling with H20-H-2 reveals a human neutrophil lifespan of 5.4 days. Blood 116: 625–627.J. PillayI. den BraberN. VrisekoopLM KwastRJ de Boer2010In vivo labeling with H20-H-2 reveals a human neutrophil lifespan of 5.4 days.Blood116625627
- 2. Brach MA, deVos S, Gruss HJ, Herrmann F (1992) Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death. Blood 80: 2920–2924.MA BrachS. deVosHJ GrussF. Herrmann1992Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death.Blood8029202924
- 3. Raza K, Scheel-Toellner D, Lee CY, Pilling D, Curnow SJ, et al. (2006) Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis. Arthritis Res Ther 8: R120.K. RazaD. Scheel-ToellnerCY LeeD. PillingSJ Curnow2006Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis.Arthritis Res Ther8R120
- 4. Leuenroth SJ, Grutkoski PS, Ayala A, Simms HH (2000) The loss of Mcl1 expression in human polymorphonuclear leukocytes promotes apoptosis. J Leukoc Biol 68: 158–166.SJ LeuenrothPS GrutkoskiA. AyalaHH Simms2000The loss of Mcl1 expression in human polymorphonuclear leukocytes promotes apoptosis.J Leukoc Biol68158166
- 5. Moulding DA, Quayle JA, Hart CA, Edwards SW (1998) Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival. Blood 92: 2495–2502.DA MouldingJA QuayleCA HartSW Edwards1998Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival.Blood9224952502
- 6. Rossi AG, Sawatzky DA, Walker A, Ward C, Sheldrake TA, et al. (2006) Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis. Nat Med 12: 1056–1064.AG RossiDA SawatzkyA. WalkerC. WardTA Sheldrake2006Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis.Nat Med1210561064
- 7. Klausen P, Bjerregaard MD, Borregaard N, Cowland JB (2004) End-stage differentiation of neutrophil granulocytes in vivo is accompanied by up-regulation of p27kip1 and down-regulation of CDK2, CDK4 and CDK6. J Leukoc Biol 75: 569–578.P. KlausenMD BjerregaardN. BorregaardJB Cowland2004End-stage differentiation of neutrophil granulocytes in vivo is accompanied by up-regulation of p27kip1 and down-regulation of CDK2, CDK4 and CDK6.J Leukoc Biol75569578
- 8. Leitch AE, Riley NA, Sheldrake TA, Festa M, Fox S, et al. (2010) The cyclin-dependent kinase inhibitor R-roscovitine down regulates Mcl-1 to overcome pro-inflammatory survival signalling and drive neutrophil apoptosis. Eur J Immunol 40: 1127–1138.AE LeitchNA RileyTA SheldrakeM. FestaS. Fox2010The cyclin-dependent kinase inhibitor R-roscovitine down regulates Mcl-1 to overcome pro-inflammatory survival signalling and drive neutrophil apoptosis.Eur J Immunol4011271138
- 9. Arris CE, Boyle FT, Calvert AH, Curtin NJ, Endicott JA, et al. (2000) Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles. J Med Chem 43: 2797–804.CE ArrisFT BoyleAH CalvertNJ CurtinJA Endicott2000Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles.J Med Chem432797804
- 10. Johnson N, Bentley J, Wang L-Z, Newell DR, Robson CN, et al. (2010) Pre-clinical evaluation of cyclin-dependent kinase 1 and 2 inhibition in anti-estrogen-sensitive and resistant breast cancer cells. Br J Cancer 102: 342–350.N. JohnsonJ. BentleyL-Z WangDR NewellCN Robson2010Pre-clinical evaluation of cyclin-dependent kinase 1 and 2 inhibition in anti-estrogen-sensitive and resistant breast cancer cells.Br J Cancer102342350
- 11. Sroka IM, Heiss EH, Havlicek L, Totzke F, Aristei Y, et al. (2010) A novel roscovitine derivative potently induces G1-phase arrest in platelet-derived growth factor –BB activated vascular smooth muscle cells. Mol Pharmacol. 77: 255–261.IM SrokaEH HeissL. HavlicekF. TotzkeY. Aristei2010A novel roscovitine derivative potently induces G1-phase arrest in platelet-derived growth factor –BB activated vascular smooth muscle cells. Mol Pharmacol.77255261
- 12. Wang S, Fischer PM (2008) Cyclin-dependent kinase 9: a key transcriptional regulator and potential drug target in oncology, virology and cardiology. Trends Pharmacol Sci. 29: 302–313.S. WangPM Fischer2008Cyclin-dependent kinase 9: a key transcriptional regulator and potential drug target in oncology, virology and cardiology. Trends Pharmacol Sci.29302313
- 13. Marshall NF, Peng J, Xie Z, Price DH (1996) Control of RNA polymerase II elongation potential by a novel carboxy-terminal domain kinase. J Biol Chem 271: 27176–27183.NF MarshallJ. PengZ. XieDH Price1996Control of RNA polymerase II elongation potential by a novel carboxy-terminal domain kinase.J Biol Chem2712717627183
- 14. Gojo I, Zhang B, Fenston RG (2002) The cyclin dependent kinase inhibitor flavopiridol induces apoptosis in multiple myeloma cells through transcriptional repression and down-regulation of Mcl-1. Clin Cancer Res 8: 3527–3538.I. GojoB. ZhangRG Fenston2002The cyclin dependent kinase inhibitor flavopiridol induces apoptosis in multiple myeloma cells through transcriptional repression and down-regulation of Mcl-1.Clin Cancer Res835273538
- 15. Rosato RR, Almenara JA, Kolla SS, Maggio SC, Coe S, et al. (2007) Mechanism and functional role of XIAP and Mcl-1 down-regulation in flavopiridol/vironostat anti-leukaemic interactions. Mol Cancer Ther 6: 692–702.RR RosatoJA AlmenaraSS KollaSC MaggioS. Coe2007Mechanism and functional role of XIAP and Mcl-1 down-regulation in flavopiridol/vironostat anti-leukaemic interactions.Mol Cancer Ther6692702
- 16. Nasreen N, Mohammed KA, Sanders KL, Hardwick J, Van Horn RD, et al. (2003) Pleural mesothelial cells modulate polymorphonuclear leukocyte apoptosis in empyema. J Clin Immunol 23: 1–10.N. NasreenKA MohammedKL SandersJ. HardwickRD Van Horn2003Pleural mesothelial cells modulate polymorphonuclear leukocyte apoptosis in empyema.J Clin Immunol23110
- 17. Derouet M, Thomas L, Cross A, Moots RJ, Edwards SW (2004) Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1. J Biol Chem 279: 26915–26921.M. DerouetL. ThomasA. CrossRJ MootsSW Edwards2004Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1.J Biol Chem2792691526921
- 18. Scheel-Toellner D, Wang KQ, Henriquez NV, Webb PR, Craddock R, et al. (2002) Cytokine mediated inhibition of apoptosis in non-transformed T cells and neutrophils can be dissociated from PKB/AKT activation. Eur J Immunol 32: 486–493.D. Scheel-ToellnerKQ WangNV HenriquezPR WebbR. Craddock2002Cytokine mediated inhibition of apoptosis in non-transformed T cells and neutrophils can be dissociated from PKB/AKT activation.Eur J Immunol32486493
- 19. Wang K, Scheel-Toellner D, Wong SH, Craddock R, Caamano J, et al. (2003) Inhibition of neutrophil apoptosis by type 1 interferon depends on cross-talk between PI-3-kinase, protein kinase C-δ and NF-κB signaling pathways. J.Immunol. 171: 1035–1041.K. WangD. Scheel-ToellnerSH WongR. CraddockJ. Caamano2003Inhibition of neutrophil apoptosis by type 1 interferon depends on cross-talk between PI-3-kinase, protein kinase C-δ and NF-κB signaling pathways. J.Immunol.17110351041
- 20. Rosales JL, Ernst JD, Hallows J, Lee KY (2004) GTP-dependent secretion from neutrophils is regulated by Cdk5. J Biol Chem. 279: 53932–53936.JL RosalesJD ErnstJ. HallowsKY Lee2004GTP-dependent secretion from neutrophils is regulated by Cdk5. J Biol Chem.2795393253936
- 21. Garriga J, Bhattacharya S, Calbo J, Marshall RM, Truongcao M, et al. (2003) CDK9 is constitutively expressed throughout the cell cycle, and its steady-state expression is independent of SKP2. Mol Cell Biol. 23: 5165–5173.J. GarrigaS. BhattacharyaJ. CalboRM MarshallM. Truongcao2003CDK9 is constitutively expressed throughout the cell cycle, and its steady-state expression is independent of SKP2. Mol Cell Biol.2351655173
- 22. O'Keeffe B, Fong Y, Chen D, Zhou S, Zhou Q (2000) Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription. J Biol Chem. 275: 279–287.B. O'KeeffeY. FongD. ChenS. ZhouQ. Zhou2000Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription. J Biol Chem.275279287
- 23. Aldridge AJ (2002) Role of the neutrophil in septic shock and the adult respiratory distress syndrome. Eur J Surgery 168: 204–214.AJ Aldridge2002Role of the neutrophil in septic shock and the adult respiratory distress syndrome.Eur J Surgery168204214
- 24. Wright HL, Moots RJ, Bucknall RC, Edwards SW (2010) Neutrophil function in inflammation and inflammatory diseases. Rheumatology 9: 1618–1631.HL WrightRJ MootsRC BucknallSW Edwards2010Neutrophil function in inflammation and inflammatory diseases.Rheumatology916181631
- 25. Carvajal RD, Tse A, Shah MA, Lefkowitz RA, Gonen M, et al. (2009) A Phase II Study of Flavopiridol (Alvocidib) in Combination with Docetaxel in Refractory, Metastatic Pancreatic Cancer. Pancreatology 9: 404–409.RD CarvajalA. TseMA ShahRA LefkowitzM. Gonen2009A Phase II Study of Flavopiridol (Alvocidib) in Combination with Docetaxel in Refractory, Metastatic Pancreatic Cancer.Pancreatology9404409
- 26. Robak T, Jamroziak K, Robak P (2009) Current and Emerging Treatments for Chronic Lymphocytic Leukaemia. Drugs 69: 2415–2449.T. RobakK. JamroziakP. Robak2009Current and Emerging Treatments for Chronic Lymphocytic Leukaemia.Drugs6924152449
- 27. Sekine C, Sugihara T, Miyake S, Hirai H, Yoshida M, et al. (2008) Successful treatment of animal models of rheumatoid arthritis with small molecule cyclin-dependent kinase inhibitors. J Immunol 180: 1954–1961.C. SekineT. SugiharaS. MiyakeH. HiraiM. Yoshida2008Successful treatment of animal models of rheumatoid arthritis with small molecule cyclin-dependent kinase inhibitors.J Immunol18019541961
- 28. Afford SC, Pongracz J, Stockley R, Crocker J, Burnett D (1992) The induction by human interleukin-6 of apoptosis in the promyelocytic cell line U937 and human neutrophils. J Biol Chem 267: 21612–21616.SC AffordJ. PongraczR. StockleyJ. CrockerD. Burnett1992The induction by human interleukin-6 of apoptosis in the promyelocytic cell line U937 and human neutrophils.J Biol Chem2672161221616
- 29. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, et al. (1989) Macrophage phagocytosis of aging neutrophils in inflammation - Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest 83: 865–875.JS SavillAH WyllieJE HensonMJ WalportPM Henson1989Macrophage phagocytosis of aging neutrophils in inflammation - Programmed cell death in the neutrophil leads to its recognition by macrophages.J Clin Invest83865875
- 30. Scheel-Toellner D, Wang K, Craddock R, Webb PR, McGettrick HM, et al. (2004) Reactive oxygen species limit neutrophil lifespan by activating death receptor signalling. Blood 104: 2557–2564.D. Scheel-ToellnerK. WangR. CraddockPR WebbHM McGettrick2004Reactive oxygen species limit neutrophil lifespan by activating death receptor signalling.Blood10425572564