cyclin-CDK Complexes Research Articles

SB202190 affects cell response to hydroxyurea-induced genotoxic stress in root meristems of Vicia faba

Genotoxic stress caused by a variety of chemical and physical agents may lead to DNA breaks and genome instability. Response to DNA damage depends on ATM/ATR sensor kinases and their downstream proteins, which arrange cell cycle checkpoints. Activation of ATM (ataxia-telangiectasia-mutated)/ATR (ATM and Rad 3-related) signaling pathway triggers cell cycle arrest (by keeping cyclin-Cdk complexes inactive), combined with gamma-phosphorylation of histone H2A.X and induction of DNA repair processes. However, genotoxic stress activates also mitogen-activated protein kinases (MAPKs) which may control the functions of checkpoint proteins both directly, by post-translational modifications, or indirectly, by regulation of their expression. Our results indicate that in root meristem cells of Vicia faba, MAP kinase signaling pathway takes part in response to hydroxyurea-induced genotoxic stress. It is shown that SB202190, an inhibitor of p38 MAP kinase, triggers PCC (premature chromosome condensation) more rapidly, but only if cell cycle checkpoints are alleviated by caffeine. Since SB202190 and, independently, caffeine reduces HU-mediated histone H4 Lys5 acetylation, it may be that there is a cooperation of MAP kinase signaling pathways and ATM/ATR-dependent checkpoints during response to genotoxic stress.

Cyclin Dependent Kinase as Significant Target for Cancer Treatment

Cyclin Dependent Kinase (CDKs) regulates cell cycle commitment and DNA synthesis. Cell division in mammalian cells is driven by protein kinase that regulates progression through the various phases of cell cycle. The activity of cyclins and their associated CDKs are frequently deranged in human cancers. For this reason, Cyclin-CDK complexes have been considered as very promising therapeutic targets in human malignancies. An obvious concern whether, blocking cyclin-CDK function would preferentially affects cancer cells but not normal and non-transformed cells. The cell cycle represents a series of tightly integrated events that allow the cell to grow and proliferate. Critical part of the cell cycle machinery is the CDK, which, when activated, provide a means for the cell to move from one phase of the cell cycle to next. The cell cycle also serves to protect the cell from DNA damage. Thus, cell cycle arrest represents a survival mechanism that provides tumour cell, to repair its own damaged DNA Thus, abrogation of cell cycle checkpoints, before DNA repair is complete can activate the apoptotic cascade leading to cell death. Misregulation of CDK is one of the most frequent alterations in human cancer. CDK are critical regulators of cell cycle progression and RNA transcription. A series of targeted agents that directly inhibit the CDKs, inhibit unrestricted cell growth, and induce growth arrest. Recent attention has also focused on these drugs as inhibitors of transcription. In this review we are summarizing that why CDK is important target for cancer chemotherapy and why finding out the best and potent kinase inhibitor is essential. Keywords: Cancer, CAK, Cell cycle, CDK, Cyclin, Kinases, Phosporylation, Threonine

Sulforaphane Induction of p21Cip1 Cyclin-dependent Kinase Inhibitor Expression Requires p53 and Sp1 Transcription Factors and Is p53-dependent

Sulforaphane (SFN) is an important cancer preventive agent derived from cruciferous vegetables. We show that SFN treatment suppresses normal human keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). SFN treatment produces a concentration-dependent increase in p21(Cip1) promoter activity via a mechanism that involves stabilization of the p53 protein leading to increased p53 binding to the p21(Cip1) promoter p53 response elements. The proximal p21(Cip1) promoter GC-rich Sp1 factor binding elements are also required, as the SFN-dependent increase is lost when these sites are mutated. SFN treatment increases Sp1 binding to these elements, and the response is enhanced in the presence of exogenous Sp1 and reduced in the presence of ΔN-Sp3. CpG island methylation alters p21(Cip1) promoter activity some systems; however, expression in SFN-treated keratinocytes does not involve changes in proximal promoter methylation. The promoter is minimally methylated, and the methylation level is not altered by SFN treatment. This study indicates that SFN increases p21(Cip1) promoter transcription via a mechanism that involves SFN-dependent stabilization of p53 and increased p53 and Sp1 binding to their respective response elements in the p21(Cip1) promoter. These results are in marked contrast to the mechanisms observed in skin cancer cell lines and suggest that SFN may protect normal keratinocytes from damage while causing cancer cells to undergo apoptosis.

Strategic Cell-Cycle Regulatory Features That Provide Mammalian Cells with Tunable G1 Length and Reversible G1 Arrest

Transitions between consecutive phases of the eukaryotic cell cycle are driven by the catalytic activity of selected sets of cyclin-dependent kinases (Cdks). Yet, their occurrence and precise timing is tightly scheduled by a variety of means including Cdk association with inhibitory/adaptor proteins (CKIs). Here we focus on the regulation of G1-phase duration by the end of which cells of multicelled organisms must decide whether to enter S phase or halt, and eventually then, differentiate, senesce or die to obey the homeostatic rules of their host. In mammalian cells, entry in and progression through G1 phase involve sequential phosphorylation and inactivation of the retinoblastoma Rb proteins, first, by cyclin D-Cdk4,6 with the help of CKIs of the Cip/Kip family and, next, by the cyclin E-Cdk2 complexes that are negatively regulated by Cip/Kip proteins. Using a dynamical modeling approach, we show that the very way how the Rb and Cip/Kip regulatory modules interact differentially with cyclin D-Cdk4,6 and cyclin E-Cdk2 provides to mammalian cells a powerful means to achieve an exquisitely-sensitive control of G1-phase duration and fully reversible G1 arrests. Consistently, corruption of either one of these two modules precludes G1 phase elongation and is able to convert G1 arrests from reversible to irreversible. This study unveils fundamental design principles of mammalian G1-phase regulation that are likely to confer to mammalian cells the ability to faithfully control the occurrence and timing of their division process in various conditions.

Abstract 4177: Synergistic role of ecdysoneless with oncogenic Ras in transformation of human mammary epithelial cells

Abstract Precisely regulated cell proliferation is essential for embryonic development as well as homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer. Thus, elucidating how the cell cycle machinery is controlled is an important area of research in cancer cell biology. A large body of evidence has established a basic paradigm of the control of cell cycle progression involving the Retinoblastoma (Rb) protein family in conjunction with the E2F family of transcription factors. During G0/G1, interaction of hypo-phosphorylated Rb proteins with E2Fs prevents the transcription of E2F target genes. Cyclin-CDK complexes generated during cell cycle progression hyper-phosphorylate Rb, leading to release of Rb from E2Fs; this allows E2F target gene transcription and cell cycle progression. We previously identified the mammalian ortholog of Drosophila ecdysoneless (Ecd) protein as a novel and essential regulator of Rb-E2F-dependent cell cycle progression. Loss of Ecd retards the separation of Rb from E2F, arrests cells at G1/S boundary and prevents cell cycle progression. Consistent with role of Ecd in cell cycle, our recent studies show Ecd is overexpressed in breast cancer cell lines as well as in ductal carcinoma in situ and infiltrating ductal carcinomas of the breast. To understand the mechanism of Ecd overexpression and its correlation with oncogenesis, we overexpressed either Ecd alone, 61L-HRas alone or both genes together in hTERT-immortalized hMECs. These transfectants were then examined for their oncogenic properties. We observed while vector infected cells do not exhibit anchorage independence, all three transfectants showed anchorage independent proliferation, particularly Ecd+Ras showed a dramatic increase in anchorage independence in comparison with either Ecd or H-Ras. Similarly, other oncogenic traits such as proliferation on matrigel, invasion and migration were dramatically increased when both genes were expressed in comparison with individual genes. Considering the known oncogenic role of Ras in human cancer and the known role of Ecd in cell cycle, these data are highly significant and present a novel synergistic pathway for mammary cell transformation. We are currently defining the molecular mechanism of this synergistic oncogenic effect. In addition, we are generating Ecd overexpressed transgenic mice to examine the role of Ecd in oncogenesis in in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4177. doi:1538-7445.AM2012-4177

Abstract 3933: ERα phosphorylation patterns at hinge domain Ser294 and Ser305 differentiate ligand-dependent from ligand-independent receptor activation

Abstract Domain-specific phosphorylation of estrogen receptor alpha (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic biomarker value. Recent attention has turned to the poorly understood ERα hinge domain in light of preliminary evidence that phosphorylation at serine 305 (pSer305) associates with poor clinical outcome and endocrine resistance. Using multiple reaction monitoring (MRM) mass spectrometry (MS), we have studied a number of ERα overexpressing human breast cancer cells lines (MCF-7, BT474, T47D) and found that robust ERα phosphorylation occurs on a neighboring hinge domain site at Ser294, but only upon ligand (estradiol, tamoxifen) stimulation of the receptor since growth factor cell stimulation (EGF, insulin, heregulin-α, forskolin) fails to induce pSer294. As well, pSer294 formation appears to be essential for full ligand induction of ERα gene transcription in experimental models. Selective inhibition of cyclin-dependent kinases (CDKs) by roscovitine or SNS-032 in the breast cancer cell lines blocks ligand induced pSer294 formation, indicating that this hinge domain phosphorylation is mediated by CDKs; likewise, cell-free studies with recombinant ERα and specific cyclin-CDK complexes reveal that Ser294 phosphorylation is most effectively induced by the transcription-regulating and cell cycle-independent kinases, CDK7 and CDK9. In contrast to this ligand specific induction of pSer294, western analysis of immunopurified ERα from MCF-7 probed with a newly available pSer305 monoclonal demonstrates that pSer305 formation occurs in response to growth factor stimulation (EGF, insulin, forskolin) but not in response to cell stimulation by ligand alone. Interestingly, co-treatment with ligand and growth factor reduces pSer294 formation relative to ligand stimulation alone while co-treatment reduces pSer305 formation relative to growth factor stimulation alone, suggesting that these two hinge phosphorylation sites not only respond reciprocally to specify different aspects of ERα function but are also antagonistically responsive to crosstalk. Unlike the better studied N-terminal domain phosphorylation sites at Ser118 and Ser167, which are phosphorylated jointly and additively upon combined ligand-dependent and ligand-independent ERα activation, the two hinge domain sites at Ser294 and Ser305 appear to respond in a reciprocal fashion differentiating ligand-dependent from ligand-independent ERα activation. Pending current development of a pSer294 monoclonal, future immunohistochemical interrogation of the ERα hinge domain phosphorylation status at Ser294 and Ser305 is expected to improve the biomarker value of this receptor by better predicting ERα overexpressing breast cancers most likely to respond to endocrine agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3933. doi:1538-7445.AM2012-3933

Short-Term Integration of Cdc25 Dynamics Controls Mitotic Entry during Drosophila Gastrulation

Cells commit to mitosis by abruptly activating the mitotic cyclin-Cdk complexes. During Drosophila gastrulation, mitosis is associated with the transcriptional activation of cdc25(string), a phosphatase that activates Cdk1. Here, we demonstrate that the switch-like entry into mitosis observed in the Drosophila embryo during the 14(th) mitotic cycle is timed by the dynamics of Cdc25(string) accumulation. The switch operates as a short-term integrator, a property that can improve the reliable control of timing of mitosis. The switch is independent of the positive feedback between Cdk1 and Cdc25(string) and of the double negative feedback between Cdk1 and Wee1. We propose that the properties of the mitotic switch are established by the out-of-equilibrium properties of the covalent modification cycle controlling Cdk1 activity. Such covalent modification cycles, triggered by transcriptional expression of the activating enzymes, might be a widespread strategy to obtain reliable and switch-like control of cell decisions.

Among B-Type Cyclins Only CLB5 and CLB6 Promote Premeiotic S Phase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cyclin Clb5 is required for premeiotic S phase, meiotic recombination, and successful progression through meiosis. Clb5 is not essential for mitotic proliferation because Clb1-Clb4 can support DNA replication in clb5 clb6 mutants. Clb1, Clb3, and Clb4 accumulate in clb5 clb6 cells during meiotic differentiation yet fail to promote premeiotic DNA replication. When expressed under the regulation of the CLB5 promoter, Clb1 and Clb3 accumulate and are active in the early stages of meiotic differentiation but cannot induce premeiotic DNA replication, suggesting that they do not target Cdk1 to the necessary substrates. The Clb5 hydrophobic patch (HP) residues are important for Clb5 function but this motif alone does not provide the specificity required for Clb5 to induce premeiotic S phase. Domain exchange experiments demonstrated that the amino terminus of Clb5 when fused to Clb3 confers upon Clb3 the ability to induce premeiotic S phase. Chimeric cyclins containing smaller regions of the Clb5 amino terminus displayed reduced ability to activate premeiotic DNA replication despite being more abundant and having greater associated histone H1 kinase activity than endogenous Clb5. These observations suggest that Clb5 has a unique ability to trigger premeiotic S phase and that the amino-terminal region of Clb5 contributes to its specificity and regulates the functions performed by the cyclin-Cdk complex.

Cyclin-Dependent Kinases Are Regulators and Effectors of Oscillations Driven by a Transcription Factor Network

During embryonic cell cycles, B-cyclin-CDKs function as the core component of an autonomous oscillator. Current models for the cell-cycle oscillator in nonembryonic cells are slightly more complex, incorporating multiple G1, S phase, and mitotic cyclin-CDK complexes. However, periodic events persist in yeast cells lacking all S phase and mitotic B-cyclin genes, challenging the assertion that cyclin-CDK complexes are essential for oscillations. These and other results led to the proposal that a network of sequentially activated transcription factors functions as an underlying cell-cycle oscillator. Here we examine the individual contributions of a transcription factor network and cyclin-CDKs to the maintenance of cell-cycle oscillations. Our findings suggest that while cyclin-CDKs are not required for oscillations, they do contribute to oscillation robustness. A model emerges in which cyclin expression (thereby, CDK activity) is entrained to an autonomous transcriptional oscillator. CDKs then modulate oscillator function and serve as effectors of the oscillator.

P27Kip1 controls cytokinesis via the regulation of citron kinase activation

p27(Kip1) (p27) acts as a tumor suppressor by inhibiting cyclin-cyclin-dependent kinase (cyclin-CDK) activity. However, mice expressing a form of p27 that is unable to bind or inhibit cyclin-CDK complexes (p27(CK-)) have increased incidence of tumor development as compared with wild-type and p27(-/-) mice, revealing an oncogenic role for p27. Here, we identified a phenotype of multinucleation and polyploidy in p27(CK-) mice not present in p27(-/-) animals, suggesting a role for p27 in G2/M that is independent of cyclin-CDK regulation. Further analysis revealed that p27(CK-) expression caused a cytokinesis and abscission defect in mouse embryonic fibroblasts. We identified the Rho effector citron kinase (citron-K) as a p27-interacting protein in vitro and in vivo and found that p27 and citron-K colocalized at the contractile ring and mid-body during telophase and cytokinesis. Moreover, overexpression of the minimal p27-binding domain of citron-K was sufficient to rescue the phenotype caused by p27(CK-). Conversely, expression of a mutant p27(CK-) unable to bind citron-K did not induce multinucleation. Finally, by binding to citron-K, p27 prevented the interaction of citron-K with its activator RhoA. Taken together, these data suggest a role for p27 during cytokinesis via the regulation of citron-K activity.

Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria

BackgroundCell proliferation is an important determinant of plant growth and development. In addition, modulation of cell-division rate is an important mechanism of plant plasticity and is key in adapting of plants to environmental conditions. One of the greatest challenges in understanding the cell cycle of flowering plants is the large families of CDKs and cyclins that have the potential to form many different complexes. However, it is largely unclear which complexes are active. In addition, there are many CDK- and cyclin-related proteins whose biological role is still unclear, i.e. whether they have indeed enzymatic activity. Thus, a biochemical characterization of these proteins is of key importance for the understanding of their function.ResultsHere we present a straightforward system to systematically express and purify active CDK-cyclin complexes from E. coli extracts. Our method relies on the concomitant production of a CDK activating kinase, which catalyzes the T-loop phosphorylation necessary for kinase activity. Taking the examples of the G1-phase cyclin CYCLIN D3;1 (CYCD3;1), the mitotic cyclin CYCLIN B1;2 (CYCB1;2) and the atypical meiotic cyclin SOLO DANCERS (SDS) in conjunction with A-, B1- and B2-type CDKs, we show that different CDKs can interact with various cyclins in vitro but only a few specific complexes have high levels of kinase activity.ConclusionsOur work shows that both the cyclin as well as the CDK partner contribute to substrate specificity in plants. These findings refine the interaction networks in cell-cycle control and pinpoint to particular complexes for modulating cell proliferation activity in breeding.

The Role of p53 and MDM2 in Head and Neck Cancer

Head and neck cancer is a complex disorder that includes mostly squamous cell carcinomas that can develop in the throat, larynx, nose, sinuses, and mouth. Etiopathogenesis is due to tobacco and alcohol consumption and to infection by human papillomavirus (HPV) type 16/18. Tumors often develop within preneoplastic fields of genetically altered cells. Most head and neck cancers result from multistep accumulation of genetic alterationsm resulting in clonal outgrowth of transformed cells. These DNA changes are caused by a variety of mechanisms like endogenous mutations and exogenous mutations. Dysregulated molecular pathway includes alterations of critical inhibitor of cyclin CDK complexes, inactivating mutations of p53 gene, and activation of oncogenes and growth factors. This paper attempts to review the role of p53 and MDM2 genetic aberrations and pathways in head and neck cancer.

Copy number defects of G1‐Cell cycle genes in neuroblastoma are frequent and correlate with high expression of E2F target genes and a poor prognosis

The tightly controlled network of cell cycle genes consists of a core of cyclin dependent kinases (CDKs) that are activated by periodically expressed cyclins. The activity of the cyclin-CDK complexes is regulated by cyclin dependent kinase inhibitors (CDKIs) and multiple signal transduction routes that converge on the cell cycle. Neuroblastoma are pediatric tumors that belong to the group of small round blue cell tumors, characterized by a fast proliferation. Here, we present high throughput analyses of cell cycle regulating genes in neuroblastoma. We analyzed a series of 82 neuroblastomas by comparative genomic hybridization arrays, single nucleotide polymorphism arrays, and Affymetrix expression arrays and analyzed the datasets in parallel with the R2 bioinformatic tool (http://r2.amc.nl). About 30% of the tumors had genomic amplifications, gains, or losses with shortest regions of overlap that suggested implication of a series of G1 cell cycle regulating genes. CCND1 (cyclin D1) and CDK4 were amplified or gained and the chromosomal regions containing the CDKN2 (INK4) group of CDKIs were frequently deleted. Cluster analysis showed that tumors with genomic aberrations in G1 regulating genes over-expressed E2F target genes, which regulate S and G2/M phase progression. These tumors have a poor prognosis. Our findings suggest that pharmacological inhibition of cell cycle genes might bear therapeutic promises for patients with high risk neuroblastoma.

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.

Retinoblastoma Protein Modulates the Inverse Relationship between Cellular Proliferation and Elastogenesis

The mechanism that leads to the inverse relationship between heightened cellular proliferation and the cessation of elastic fibers production, observed during formation of the arterial occlusions and dermal scars, is not fully understood. Because the retinoblastoma protein (Rb), responsible for cell cycle initiation, has also been implicated in insulin-like growth factor-I-mediated signaling stimulating elastin gene activation, we explored whether differential phosphorylation of Rb by various cyclin·cyclin-dependent kinase complexes would be responsible for promoting either elastogenic or pro-proliferative signals. We first tested cultures of dermal fibroblasts derived from Costello syndrome patients, in which heightened proliferation driven by mutated oncogenic H-Ras coincides with inhibition of elastogenesis. We found that Costello syndrome fibroblasts display elevated level of Rb phosphorylation on serine 780 (Ser(P)-780-Rb) and that pharmacological inhibition of Ras with radicicol, Mek/Erk with PD98059, or cyclin-dependent kinase 4 with PD0332991 not only leads to down-regulation of Ser(P)-780-Rb levels but also enhances Rb phosphorylation on threonine-821 (Thr(P)-821-Rb), which coincides with the recovery of elastin production. Then we demonstrated that treatment of normal skin fibroblasts with the pro-proliferative PDGF BB also up-regulates Ser(P)-780-Rb levels, but treatment with the pro-elastogenic insulin-like growth factor-I activates cyclinE-cdk2 complex to phosphorylate Rb on Thr-821. Importantly, we have established that elevation of Thr(P)-821-Rb promotes Rb binding to the Sp1 transcription factor and that successive binding of the Rb-Sp1 complex to the retinoblastoma control element within the elastin gene promoter stimulates tropoelastin transcription. In summary, we provide novel insight into the role of Rb in mediating the inverse relationship between elastogenesis and cellular proliferation.

Structure of a cyclin-dependent kinase from Giardia lamblia.

Giardia lamblia is the etiologic agent of giardiasis, a water-borne infection that is prevalent throughout the world. The need for new therapeutics for the treatment of giardiasis is of paramount importance. Owing to the ubiquitous nature of kinases and their vital importance in organisms, they are potential drug targets. In this paper, the first structure of a cyclin-dependent kinase (CDK) from G. lamblia (GlCDK; UniProt A8BZ95) is presented. CDKs are cell-cycle-associated kinases that are actively being pursued as targets for anticancer drugs as well as for antiparasitic chemotherapy. Generally, a CDK forms a complex with its associated cyclin. This CDK-cyclin complex is active and acts as a serine/threonine protein kinase. Typically, CDKs are responsible for the transition to the next phase of the cell cycle. Although the structure of GlCDK with its associated cyclin was not solved, the 1.85 Å resolution structure of apo GlCDK and a 2.0 Å resolution structure of GlCDK in complex with adenosine monophosphate are presented and the structural differences from the orthologous human CDK2 and CDK3 are discussed.

Peptides or Small Molecules? Different Approaches to Develop More Effective CDK Inhibitors

Cell cycle regulation involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. The molecular events that control cell cycle are ordered and directional. Cyclins and cyclin-dependent kinases (CDKs), determine cell progression through the cycle ensuring the orderly coordination of cellular events. Alterations of cell cycle controllers are among the main causes of cancer onset. In the past decades many efforts have been made to develop kinase inhibitors that are able to modulate cyclin and CDK complexes, either by mimicking the function of natural CDK inhibitors, such as p21, p16 and p27, or by modulating the cyclin-CDK complexes or their targets directly. The great debate is whether to use peptides or small molecules. Peptides are more selective being derived by the linear protein sequences, indeed they should mimic the catalytic or the regulatory subunits of the cell cycle controller complexes, but on the other side they usually present poorer pharmacokinetic characteristics. In contrast, small molecules have better pharmacokinetic features but lower specificity because many protein kinases show high sequence similarity within the active site. The purpose of this review article is to provide an overview of the main classes of CDK inhibitors focusing on structure-activity relationship (SAR) studies and discussing the pharmacological and therapeutic implications.

Abstract 2938: Inhibition of PI3K/AKT and MEK/ERK pathways act independently on p27 upregulation induced by downregulation of cathepsin B and uPAR in glioma

Abstract Cathepsin B and urokinase plasminogen activator receptor (uPAR) are overexpressed in gliomas. Deregulation of the G1 phase cell cycle machinery is a common feature of cancers. The abundance of cyclin-dependent kinase inhibitor p27Kip1 (p27) during the cell cycle determines whether cells will proliferate or become quiescent. In the present study, we carried out transfections using shRNA against cathepsin B and uPAR. We assessed cell cycle arrest using FACS analysis and used Western blot analysis to detect expression of ERK, AKT, p27, cyclin E, cyclin D, CDK4, CDK2, EGFR, β1 integrins, and the pocket proteins (p-Rb, p107 and p130). Specific inhibitors like Wortmanin (10 µM) and U0126 (10 µM) were used to further determine the roles of the AKT and ERK pathways. We used immunoprecipitation analysis to detect the respective interactions between EGFR and β1, p27 and CDK4, and p27 and CDK2. CHIP analysis was used to check for the recruitment of transcription factors FOXO3a, E2F1 and AP-1 to the p27 promoter. The results of the present study show that shRNA treatment efficiently downregulated expression of cathepsin B and uPAR and induced G0/G1 arrest through upregulation of p27. Downregulation of both p-ERK and p-AKT downstream of EGFR and β1 are involved, but independently, in p27 upregulation. Multiple transcription factors (namely, AP-1, E2F1 and FOXO3a) were involved in upregulation of p27 as determined by luciferase expression under the influence of promoter regions to which these transcription factors bind individually. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, the reduction of p-ERK and AP-1, the increased expression of E2F1 and FOXO3a, and the reduced association of cyclin D-CDK4 and cyclin E-CDK2 complexes contributed to p27 upregulation as well as G0/G1 arrest induced by downregulation of cathepsin B and uPAR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2011-2938

Escape from p21-mediated Oncogene-induced Senescence Leads to Cell Dedifferentiation and Dependence on Anti-apoptotic Bcl-xL and MCL1 Proteins

Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.

The mechanism of phenoptosis: 2. The Hayflick limit is caused by programmed decrease of the bioenergetics level

This paper continues an early attempt at substantiation of the programmed aging mechanism (phenoptosis). The concept underlying this mechanism is that life represents many interrelated physical and chemical processes propelled by bioenergetics. The gradual programmed decrease of the bioenergetics level causes slow and coordinated weakening of all physiological functions, i.e., aging. For a convincing substantiation of such a mechanism, it is necessary to show how the bioenergetics decrease causes the basic harmful processes accompanying aging. It has been shown earlier that the age-dependent decrease of the bioenergetics level causes an increase of reactive oxygen production by mitochondria and decrease of the overall level of protein synthesis. Evidence that termination of cell proliferation after a certain number of doublings (the Hayflick limit) is also caused by the age-related decrease of the bioenergetics level is presented in this paper. Old cells stop dividing because decrease of the bioenergetics level below a certain critical level deprives a cell of its ability to pass the G1-phase restriction point of the proliferative cycle. The cyclin-dependent kinase inhibitor p27 prevents a cell (except embryonic and cancer cells) from passing through this critical point. During division of normal somatic cells, p27 is removed by the active cyclin E-Cdk2 complex in response to mitogen stimulation. Interaction of p27 with the cyclin E-Cdk2 complex can have two consequences. At the normal physiological level of bioenergetics, the cyclin E-Cdk2 phosphorylates p27. After that the latter is destroyed by proteolytic enzymes and the cell enters the S-phase. If the bioenergetics level decreases below a certain level, the cyclin E-Cdk2 becomes a target for p27. As a result the inhibitor elimination stops and a restriction point becomes closed—a cell enters an irreversible proliferative rest.