Cyclin-dependent Kinase Activity Research Articles

Functionally active cyclin-dependent kinase 9 is essential for porcine reproductive and respiratory syndrome virus subgenomic RNA synthesis

Cyclin-dependent kinase 9 (CDK9) is a key regulator of RNA-polymerase II and a candidate therapeutic target for various virus infections such as respiratory syncytial virus, herpes simplex virus, human adenovirus, human cytomegalovirus, hepatitis virus B, and human papillomavirus. We employed CDK9-IN-1, a selective CDK9 inhibitor, to investigate the role of CDK9 in porcine reproductive and respiratory syndrome virus (PRRSV) infection. CDK9-IN-1 dose-dependently reduced PRRSV replication without cytotoxicity in the infected cells. The antiviral activity of CDK9-IN-1 was further confirmed by evaluating the effects of lentivirus-mediated CDK9 knockdown or CDK9 overexpression on PRRSV infection. Briefly, the depletion of CDK9 significantly inhibited viral replication, while the overexpression of CDK9 promoted viral replication. PRRSV infection also enhanced the nuclear export of CDK9 without affecting CDK9 protein expression. Viral replication cycle analyses further revealed that functionally active CDK9 in the cytosol advanced viral subgenomic RNA synthesis. Collectively, our data illustrated that CDK9 was a new host factor that was involved in PRRSV subgenomic RNA synthesis, and CDK9 inhibitor, CDK9-IN-1 was a promising antiviral candidate for PRRSV infection.

Lipopolysaccharide-induced inflammation in human peritoneal mesothelial cells is controlled by ERK1/2-CDK5-PPARγ axis.

BackgroundPeritonitis is a common complication in which the peritoneum becomes inflamed. Peroxisome proliferator-activated receptor (PPAR)γ agonists and extracellular signal-regulated kinases 1/2 (ERK1/2) inactivation have been found to restore damage caused by lipopolysaccharide-induced (LPS) inflammation. This study aimed to investigate the association between PPARγ and ERK1/2 in LPS-induced inflammation in peritonitis.MethodsHuman peritoneal mesothelial cells were maintained in Dulbecco’s Modified Eagle Medium and treated with LPS under a series of different concentrations and treatment times. Cellular interleukins-1βeta (IL-1β), cellular interleukins-6 (IL-6), cellular interleukins-12 (IL-12) were measured by enzyme-linked immunosorbent assay (ELISA) assay. Expression or activation of cyclin-dependent kinase (CDK)5, ERK1/2, and PPARγ was detected using quantitative real-time PCR and/or western blot.ResultsLPS induced dose- and time-dependent increments in the cellular IL-1β, IL-6, and IL-12 contents, cyclin-dependent kinase 5 (CDK5) expression, and PPARγSer273 phosphorylation. Treatment with 1 µg/mL LPS for 12 hours was the optimal experimental design for inflammation stimulation. The concentration of LPS over 1 µg/mL or treatment more than 12 hours reduced the inflammatory status. LPS stimulation also activated ERK1/2 and increased its interaction with CDK5. Further, ERK1/2 inhibition by AZD0364 prevented IL-1β, IL-6, IL-12, and CDK5 expression, as well as activation of ERK1/2 and phosphorylation of PPARγ, induced by LPS. Knockdown of CDK5 using its siRNA caused similar changes as AZD0364, minus ERK1/2 inactivation.ConclusionsOur results suggested that LPS-induced inflammation in human peritoneal mesothelial cells can be partly suppressed by inhibiting the ERK1/2/CDK5/PPARγ axis.

Cyclin-dependent kinase inhibitors (CDKIs) and the DNA damage response: The link between signaling pathways and cancer

At the cellular level, DNA repair mechanisms are crucial in maintaining both genomic integrity and stability. DNA damage appears to be a central culprit in tumor onset and progression. Cyclin-dependent kinases (CDKs) and their regulatory partners coordinate the cell cycle progression. Aberrant CDK activity has been linked to a variety of cancers through deregulation of cell-cycle control. Besides DNA damaging agents and chromosome instability (CIN), disruptions in the levels of cell cycle regulators including cyclin-dependent kinase inhibitors (CDKIs) would result in unscheduled proliferation and cell division. The INK4 and Cip/Kip (CDK interacting protein/kinase inhibitor protein) family of CDKI proteins are involved in cell cycle regulation, transcription regulation, apoptosis, and cell migration. A thorough understanding of how these CDKIs regulate the DNA damage response through multiple signaling pathways may provide an opportunity to design efficient treatment strategies to inhibit carcinogenesis.

The Less Known Cyclins—Uncovered

Cyclins belong to a group of proteins that are cyclically produced and destructed in a cell. Cyclins are a family of proteins that are a key component of the cell cycle regulating system, which level of expression depends on the phase of the cycle. Cyclins regulate the activity of cyclin-dependent kinases (Cdk), thanks to which they influence the length of individual phases of the cell cycle and also determine whether the cell can enter the next life stage. Proper expression of cyclins plays an important role in processes such as proliferation, transcription, DNA repair and cell differentiation. However, dysregulation of their expression is one of the most important disorders leading to the development of different types of cancer, which suggests that cyclins can be defined as a prognostic marker. Currently, we may distinguish >10 members of the cyclins family participating in the division of human cells. The group of less known cyclins includes C, F, G, H, I, J, K, L, M, O, T and Y cyclins. The present report demonstrates the current state of knowledge considering less known cyclins and their role in normal and cancer cells.

New Insights into CDK Regulators: Novel Opportunities for Cancer Therapy.

Cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), control the transition between different phases of the cell cycle. CDK/cyclin activity is regulated by CDK inhibitors (CKIs), currently comprising the CDK-interacting protein/kinase inhibitory protein (CIP/KIP) family and the inhibitor of kinase (INK) family. Recent studies have identified a third group of CKIs, called ribosomal protein-inhibiting CDKs (RPICs). RPICs were discovered in the context of cellular senescence, a stable cell cycle arrest with tumor-suppressing abilities. RPICs accumulate in the nonribosomal fraction of senescent cells due to a decrease in rRNA biogenesis. Accordingly, RPICs are often downregulated in human cancers together with other ribosomal proteins, the tumor-suppressor functions of which are still under study. In this review, we discuss unique therapies that have been developed to target CDK activity in the context of cancer treatment or senescence-associated pathologies, providing novel tools for precision medicine.

Hyperactivation of cyclin A-CDK induces centrosome overduplication and chromosome tetraploidization in mouse cells

Mammalian cyclin A-CDK (cyclin-dependent kinase) activity during mitotic exit is regulated by two redundant pathways, cyclin degradation and CDK inhibitors (CKIs). Ectopic expression of a destruction box-truncated (thereby stabilized) mutant of cyclin A in the mouse embryonic fibroblasts nullizygous for three CKIs (p21, p27, and p107) results in constitutive activation (“hyperactivation”) of cyclin A-CDK and induces rapid tetraploidization, suggesting loss of the two redundant pathways causes genomic instability. To elucidate the mechanism underlying teraploidization by hyperactive cyclin A-CDK, we first examined if the induction of tetraploidization depends on specific cell cycle stage(s). Arresting the cell cycle at either S phase or M phase blocked the induction of tetraploidization, which was restored by subsequent release from the arrest. These results suggest that both S- and M-phase progressions are necessary for the tetraploidization by hyperactive cyclin A-CDK and that the tetraploidization is not caused by chromosome endoreduplication but by mitotic failure. We also observed that the induction of tetraploidization is associated with excessive duplication of centrosomes, which was suppressed by S-phase but not M-phase block, suggesting that hyperactive cyclin A-CDK promotes centrosome overduplication during S phase. Time-lapse microscopy revealed that hyperactive cyclin A-CDK can lead cells to bypass cell division and enter pseudo-G1 state. These observations implicate that hyperactive cyclin A-CDK causes centrosome overduplication, which leads to mitotic slippage and subsequent tetraploidization.

BAF57/SMARCE1 Interacting with Splicing Factor SRSF1 Regulates Mechanical Stress-Induced Alternative Splicing of Cyclin D1.

Background: Cyclin D1 regulates cyclin-dependent protein kinase activity of the cell cycle, and cyclin D1 alternative splicing generates a cyclin D1b isoform, acting as a mediator of aberrant cellular proliferation. As alternative splicing processes are sensitive to mechanical stimuli, whether the alternative splicing of cyclin D1 is regulated by mechanical stress and what kinds of factors may act as the regulator of mechano-induced alternative splicing remain unknown. Methods: The alternative splicing of Cyclin D1 was examined using reverse transcription polymerase chain reaction (RT-PCR) in osteoblast cell lines and keratinocyte cells loaded by a cyclic stretch. The expression of splicing factors and switching defective/sucrose non-fermenting (SWI/SNF) complex subunits were detected in stretched cells using real-time quantitative PCR (RT-qPCR). The protein interaction was tested by co-immunoprecipitation assay (Co-IP). Results: Cyclin D1 expression decreased with its splice variant upregulated in stretched cells. Serine/arginine-rich splicing factor 1 (SRSF1) and SWI/SNF complex subunit Brahma-related gene-1-associated factor 57 (BAF57), also named SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1 (SMARCE1), could respond to mechanical stimuli. Overexpression and knockdown experiments indicated the BAF57/SMARCE1 is probably a critical factor regulating the alternative splicing of cyclin D1. Co-IP showed an interaction between BAF57/SMARCE1 and SRSF1, implying a possible underlying mechanism of the regulator role of BAF57/SMARCE1 in the splicing process of cyclin D1. Conclusions: The splicing factor SRSF1 and BAF57/SMARCE1 are possibly responsible for the mechanical stress-induced alternative splicing of cyclin D1.

Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

Pyrazole[3,4-d]pyrimidine derivatives loaded into halloysite as potential CDK inhibitors

Uncontrolled cell proliferation is a hallmark of cancer as a result of rapid and deregulated progression through the cell cycle. The inhibition of cyclin-dependent kinases (CDKs) activities is a promising therapeutic strategy to block cell cycle of tumor cells. In this work we reported a new example of nanocomposites based on halloysite nanotubes (HNTs)/pyrazolo[3,4-d]pyrimidine derivatives (Si306 and Si113) as anticancer agents and CDK inhibitors. HNTs/Si306 and HNTs/Si113 nanocomposites were synthesized and characterized. The release kinetics were also investigated. Antitumoral activity was evaluated on three cancer cell lines (HeLa, MDA-MB-231 and HCT116) and the effects on cell cycle arrest in HCT116 cells were evaluated. Finally, molecular dynamics simulations were performed of the complexes between Si113 or Si306 and the active site of both CDK 1 and 2.

The Synaptic Dysregulation in Adolescent Rats Exposed to Maternal Immune Activation.

Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders in offspring, but the pathomechanism is largely unknown. The aim of our study was to analyse the molecular mechanisms contributing to synaptic alterations in hippocampi of adolescent rats exposed prenatally to MIA. MIA was evoked in pregnant female rats by i.p. administration of lipopolysaccharide at gestation day 9.5. Hippocampi of offspring (52–53-days-old rats) were analysed using transmission electron microscopy (TEM), qPCR and Western blotting. Moreover, mitochondrial membrane potential, activity of respiratory complexes, and changes in glutathione system were measured. It was found that MIA induced changes in hippocampi morphology, especially in the ultrastructure of synapses, including synaptic mitochondria, which were accompanied by impairment of mitochondrial electron transport chain and decreased mitochondrial membrane potential. These phenomena were in agreement with increased generation of reactive oxygen species, which was evidenced by a decreased reduced/oxidised glutathione ratio and an increased level of dichlorofluorescein (DCF) oxidation. Activation of cyclin-dependent kinase 5, and phosphorylation of glycogen synthase kinase 3β on Ser9 occurred, leading to its inhibition and, accordingly, to hypophosphorylation of microtubule associated protein tau (MAPT). Abnormal phosphorylation and dysfunction of MAPT, the manager of the neuronal cytoskeleton, harmonised with changes in synaptic proteins. In conclusion, this is the first study demonstrating widespread synaptic changes in hippocampi of adolescent offspring prenatally exposed to MIA.

SUMOylation mediates CtIP's functions in DNA end resection and replication fork protection.

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP’s function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP’s carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.

Partial Inhibition of Mitochondrial Complex I Reduces Tau Pathology and Improves Energy Homeostasis and Synaptic Function in 3xTg-AD Mice.

Background:Accumulation of hyperphosphorylated tau (pTau) protein is associated with synaptic dysfunction in Alzheimer’s disease (AD). We previously demonstrated that neuroprotection in familial mouse models of AD could be achieved by targeting mitochondria complex I (MCI) and activating the adaptive stress response. Efficacy of this strategy on pTau-related pathology remained unknown.Objective:To investigate the effect of specific MCI inhibitor tricyclic pyrone compound CP2 on levels of human pTau, memory function, long term potentiation (LTP), and energy homeostasis in 18-month-old 3xTg-AD mice and explore the potential mechanisms.Methods:CP2 was administered to male and female 3xTg-AD mice from 3.5–18 months of age. Cognitive function was assessed using the Morris water maze. Glucose metabolism was measured in periphery using a glucose tolerance test and in the brain using fluorodeoxyglucose F18 positron-emission tomography (FDG-PET). LTP was evaluated using electrophysiology in the hippocampus. The expression of key proteins associated with neuroprotective mechanisms were assessed by western blotting.Results:Chronic CP2 treatment restored synaptic activity in female 3xTg-AD mice; cognitive function, levels of synaptic proteins, glucose metabolism, and energy homeostasis were improved in male and female 3xTg-AD mice. Significant reduction of human pTau in the brain was associated with increased activity of protein phosphatase of type 2A (PP2A), and reduced activity of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β).Conclusion:CP2 treatment protected against synaptic dysfunction and memory impairment in symptomatic 3xTg-AD mice, and reduced levels of human pTau, indicating that targeting mitochondria with small molecule specific MCI inhibitors represents a promising strategy for treating AD.

KIFC1 overexpression promotes prostate cancer cell survival and proliferation in vitro by clustering of amplified centrosomes via interaction with Centrin 2

Mitotic kinesin KIFC1 plays critical roles in mitosis by regulating the spindle length, pole formation, and known for clustering extra centrosomes in cancer cells. Centrosome clustering is associated with the survival of cancer cells, but this phenomenon remains obscure in prostate cancer (PCa). The present study demonstrated that PCa cells showed centrosome amplification and clustering during interphase and mitosis, respectively. KIFC1 is highly expressed in PCa cells and tumor tissues of prostatic adenocarcinoma (PAC) patients. Up-regulation of KIFC1 facilitated the PCa cell survival in vitro by ensuring bipolar mitosis through clustering the multiple centrosomes, suggesting centrosome clustering could be a leading cause of prostate carcinogenesis. Conversely, the silencing of KIFC1 resulted in normal centrosome number or multipolar mitosis by inhibiting the clustering of amplified centrosomes in PCa cells. Besides, knockdown of KIFC1 by RNAi in PCa cells reduced cancer cell survival, and proliferation. KIFC1 interacted with centrosome structural protein Centrin 2 in clustering of amplified centrosomes in PCa cells to ensure the bipolar mitotic spindle formation. Knockdown of Centrin 2 in PCa cells inhibited the centrosome amplification and clustering. Moreover, up-regulated KIFC1 promotes PCa cell proliferation via progression of cell cycle possibly through aberrant activation of cyclin dependent kinase 1(Cdk1). Therefore, KIFC1 can be a prognostic marker and therapeutic target of PCa for inhibiting the cancer cell proliferation.

Real-Time Monitoring of APC /C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts.

The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C-Cdk1 activating the APC/C and the APC/C inactivating Cdk1-constitutes the core of the biochemical cell cycle oscillator.Since its discovery three decades ago, the mechanisms of APC /C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC /C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC /C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC /C activity in Xenopus laevis egg extracts using APC /C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC /C mutants or investigate other mechanisms of APC /C regulation.

Fluorescent Peptide Biosensors for Probing CDK Kinase Activity in Cell Extracts.

Fluorescent biosensors can report on the relative abundance, activity, or conformation of biomolecules and analytes through changes in fluorescence emission. A wide variety of genetically-encoded and synthetic biosensors have been developed to monitor protein kinase activity. We have focused on the design, engineering and characterization of fluorescent peptide biosensors of cyclin-dependent kinases (CDKs) that constitute attractive cancer biomarkers and pharmacological targets. In this chapter, we describe the CDKACT fluorescent peptide biosensor technology and its application to assess the relative kinase activity of CDKs in vitro, either using recombinant proteins or cell extracts as a more complex source of kinase. This technology offers a straightforward means of comparing CDK activity in different cell lines and evaluating the specific impact of treatments intended to target kinase activity in a physiologically relevant environment.

Ongoing replication forks delay the nuclear envelope breakdown upon mitotic entry

DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S phase of the cell cycle. If these stalled forks persist into the M phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5′-triphosphate–induced stalled forks were able to restart with the addition of excess dCTP during early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after the NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of the NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase activities because of which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

Visualizing the metazoan proliferation-quiescence decision in vivo.

Cell proliferation and quiescence are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these key events of the cell cycle in Caenorhabditis elegans and zebrafish through live-cell imaging. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from quiescent cells in G0, revealing a possible commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. Finally, we derive a predictive model of future proliferation behavior in C. elegans based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell-cycle control in a wide-range of developmental contexts.

Flavopiridol Induces Apoptosis of Normal Lymphoid Cells, Causes Immunosuppression, and Has Potent Antitumor Activity In Vivo Against Human Leukemia and Lymphoma Xenografts

AbstractFlavopiridol is a novel semisynthetic flavone derivative of the alkaloid rohitukine. Flavopiridol is known to inhibit potently the activity of multiple cyclin-dependent kinases. We have assessed its effects on normal and malignant cells in preclinical animal models of localized and disseminated human hematopoietic neoplasms. Flavopiridol, when administered as daily bolus intravenous (IV) injections, produced selective apoptosis of cells in the thymus, spleen, and lymph nodes, resulting in atrophy of these organs. With the exception of the intestinal crypts, apoptosis or tissue damage was absent in all other organs investigated (kidneys, liver, lungs, bone/bone marrow, muscle, and heart). Flavopiridol had a marked apoptotic effect documented by DNA nick-end labeling, or DNA agarose gels in xenografts of human hematopoietic tumors HL-60, SUDHL-4, and Nalm/6. After treatment with 7.5 mg/kg flavopiridol bolus IV or intraperitoneal on each of 5 consecutive days, 11 out of 12 advanced stage subcutaneous (s.c.) human HL-60 xenografts underwent complete regressions, and animals remained disease-free several months after one course of flavopiridol treatment. SUDHL-4 s.c. lymphomas treated with flavopiridol at 7.5 mg/kg bolus IV for 5 days underwent either major (two out of eight mice) or complete (four out of eight mice) regression, with two animals remaining disease-free for more than 60 days. The overall growth delay was 73.2%. The acquired immunodeficiency syndrome–associated lymphoma AS283 showed no significant response when flavopiridol was used in advanced s.c. tumors, but when treatment was initiated in early stages, there was a complete regression of the early tumors, and a significant overall growth delay (>84%). When flavopiridol was used in severe combined immunodeficient mice bearing disseminated human acute lymphoblastic leukemia Nalm/6 cells, there was 15-day prolongation in survival (P = .0089). We conclude that flavopiridol greatly influences apoptosis in both normal and malignant hematopoietic tissues. This activity was manifested in our study as a potent antileukemia or antilymphoma effect in human tumor xenografts, which was dose and schedule dependent. These findings provide compelling evidence for the use of flavopiridol in human hematologic malignancies.

Cyclin F Downregulation Affects Epithelial-Mesenchymal Transition Increasing Proliferation and Migration of the A-375 Melanoma Cell Line.

BackgroundCyclins are well-known cell cycle regulators. The activation of cyclin-dependent kinases by cyclins allows orchestration of the complicated cell cycle machinery and drives the cell from the G1 phase to the end of the mitotic phase. In recent years, it has become evident that cyclins are involved in processes beyond the cell cycle. Cyclin F does not activate CDKs but forms part of the Skp1-Cul1-F-box (SCF) complex where it is responsible for protein target recognition and subsequent degradation in a proteasome-dependent manner.ResultsHere, we report that the downregulation of cyclin F in the A-375 melanoma cell line increases cell viability and colony formation in a cell cycle independent manner. Lower levels of cyclin F do not appear to affect the cell cycle, based on flow cytometry measuring BrdU incorporation and propidium iodide staining. By means of immunofluorescence staining and Western blot analysis, we observed changes in cell morphology-related markers which suggested ongoing epithelial-mesenchymal transition (EMT) in response to cyclin F downregulation. Increases in vimentin and N-cadherin protein levels, decreases in levels of epithelial markers such as ZO-1, along with changes in morphology to a spindle-like shape with the appearance of actin stress fibers, are all hallmarks of EMT. These changes are associated with increased invasive and migratory potential, based on 2D migration assays. Moreover, we observe an increase in RhoABC, talin and paxillin levels, the proteins involved in controlling cell signaling and motility. Lastly, upon knocking down cyclin F expression, we observed a decrease in thrombospondin-1 expression, suggesting a role of cyclin F in angiogenesis.ConclusionCyclin F depletion induces proliferation and EMT processes in the A-375 melanoma model.

Angelika Amon.

Angelika Amon.