Degradation Of Cell Cycle Regulators Research Articles

Efficient terminal erythroid differentiation requires the APC/C cofactor Cdh1 to limit replicative stress in erythroblasts

The APC/C-Cdh1 ubiquitin ligase complex drives proteosomal degradation of cell cycle regulators and other cellular proteins during the G1 phase of the cycle. The complex serves as an important modulator of the G1/S transition and prevents premature entry into S phase, genomic instability, and tumor development. Additionally, mounting evidence supports a role for this complex in cell differentiation, but its relevance in erythropoiesis has not been addressed so far. Here we show, using mouse models of Cdh1 deletion, that APC/C-Cdh1 activity is required for efficient terminal erythroid differentiation during fetal development as well as postnatally. Consistently, Cdh1 ablation leads to mild but persistent anemia from birth to adulthood. Interestingly, loss of Cdh1 seems to affect both, steady-state and stress erythropoiesis. Detailed analysis of Cdh1-deficient erythroid populations revealed accumulation of DNA damage in maturing erythroblasts and signs of delayed G2/M transition. Moreover, through direct assessment of replication dynamics in fetal liver cells, we uncovered slow fork movement and increased origin usage in the absence of Cdh1, strongly suggesting replicative stress to be the underlying cause of DNA lesions and cell cycle delays in erythroblasts devoid of Cdh1. In turn, these alterations would restrain full maturation of erythroblasts into reticulocytes and reduce the output of functional erythrocytes, leading to anemia. Our results further highlight the relevance of APC/C-Cdh1 activity for terminal differentiation and underscore the need for precise control of replication dynamics for efficient supply of red blood cells.

SCF Ligases and Their Functions in Oogenesis and Embryogenesis-Summary of the Most Important Findings throughout the Animal Kingdom.

SCF-dependent proteolysis was first discovered via genetic screening of budding yeast almost 25 years ago. In recent years, more and more functions of SCF (Skp1-Cullin 1-F-box) ligases have been described, and we can expect the number of studies on this topic to increase. SCF ligases, which are E3 ubiquitin multi-protein enzymes, catalyse protein ubiquitination and thus allow protein degradation mediated by the 26S proteasome. They play a crucial role in the degradation of cell cycle regulators, regulation of the DNA repair and centrosome cycle and play an important role in several diseases. SCF ligases seem to be needed during all phases of development, from oocyte formation through fertilization, activation of the embryonic genome to embryo implantation. In this review, we summarize known data on SCF ligase-mediated degradation during oogenesis and embryogenesis. In particular, SCFβTrCP and SCFSEL-10/FBXW7 are among the most important and best researched ligases during early development. SCFβTrCP is crucial for the oogenesis of Xenopus and mouse and also in Xenopus and Drosophila embryogenesis. SCFSEL-10/FBXW7 participates in the degradation of several RNA-binding proteins and thereby affects the regulation of gene expression during the meiosis of C. elegans. Nevertheless, a large number of SCF ligases that are primarily involved in embryogenesis remain to be elucidated.

RSK Inhibition Induces Metaphase Arrest and Apoptosis in Acute Myeloid Leukemia Cells

The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in several cancers, although its oncogenic mechanism has not been well characterized. To examine the role of RSK in acute myeloid leukemia (AML), we analyzed apoptosis, DNA-damage, and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment (5 µM, 12 h) of HL-60 and KG-1 cells increased the G2/M population and induced apoptosis. Previously, RSK has been shown to activate CDC2 for G2/M progression by dephosphorylating the negative regulatory Tyr14 through activation of CDC25 or inhibition of MYT1. Phosphorylation of CDC2 at Tyr14 was increased transiently during G2 phase following treatment with BI-D1870, while phosphorylation of the positive regulatory Ser198 of CDC25C was inhibited in G2 and mitotic populations. However, BI-D1870 treatment did not decrease CDC2/Cyclin B kinase activity, suggesting that inhibition of CDC2 is not a critical regulatory mechanism in BI-D1870-induced mitotic arrest. The mitotic population was further characterized by evaluation of expression levels of Cyclin A and Cyclin B with flow cytometry. Approximately 70% of mitotic cells accumulated in Cyclin A-negative/Cyclin B-high metaphase at 12 hours after 5 µM BI-D1870 treatment. Metaphase arrest was confirmed using a thymidine/nocodazole block to achieve synchronization at metaphase. Our results show that the metaphase/anaphase transition is significantly stalled by RSK inhibition as assessed by degradation of Cyclin B and Securin following release from mitotic arrest (Metaphase cells in mitotic population (%), control vs. BI-D1870, 0 h post-release: 80.53 ± 0.09% vs. 88.03 ± 0.67%; 2 h post-release: 56.27 ± 1.42% vs. 83.37 ± 0.32%; 4 h post-release: 23.53 ± 0.52% vs. 62.90 ± 0.62%, mean ± SEM, n=3, p< 0.01). We also assessed the efficacy of combination treatment of BI-D1870 with standard AML chemotherapeutic drugs cytarabine and daunorubicin. The combination of BI-D1870 with cytarabine or daunorubicin in HL-60 cells showed an additive effect. Furthermore, BI-D1870 induced apoptosis and metaphase arrest in primary AML cells and significantly suppressed the CD34+ subpopulation containing leukemia stem cells. Mitotic progression is controlled by the sequential degradation of cell cycle regulators including Cyclin A, Cyclin B, and Securin by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Since proper association of CDC20 with APC/C is critical to destroy Cyclin B and Securin for metaphase/anaphase progression, we examined the effect of BI-D1870 on the complex formation of CDC20 with APC/C. Co-IP experiments showed that treatment of BI-D1870 impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to APC/C, preventing metaphase/anaphase progression. These data show that BI-D1870 inhibits metaphase/anaphase progression by inactivating APC/C, resulting in apoptosis of AML cells. We are currently studying the effects of RSK knockdown on AML cells. In summary, our study demonstrates that RSK plays an important role in maintaining AML cell survival and proliferation, and RSK inhibitors could be used therapeutically to treat AML by inducing apoptosis and cell cycle arrest through the inhibition of APC/C in AML cells. DisclosuresNo relevant conflicts of interest to declare.

Heat Shock Cognate 70 Functions as A Chaperone for the Stability of Kinetochore Protein CENP-N in Holocentric Insect Silkworms.

The centromere, in which kinetochore proteins are assembled, plays an important role in the accurate congression and segregation of chromosomes during cell mitosis. Although the function of the centromere and kinetochore is conserved from monocentric to holocentric, the DNA sequences of the centromere and components of the kinetochore are varied among different species. Given the lack of core centromere protein A (CENP-A) and CENP-C in the lepidopteran silkworm Bombyx mori, which possesses holocentric chromosomes, here we investigated the role of CENP-N, another important member of the centromere protein family essential for kinetochore assembly. For the first time, cellular localization and RNA interference against CENP-N have confirmed its kinetochore function in silkworms. To gain further insights into the regulation of CENP-N in the centromere, we analyzed the affinity-purified complex of CENP-N by mass spectrometry and identified 142 interacting proteins. Among these factors, we found that the chaperone protein heat shock cognate 70 (HSC70) is able to regulate the stability of CENP-N by prohibiting ubiquitin–proteasome pathway, indicating that HSC70 could control cell cycle-regulated degradation of CENP-N at centromeres. Altogether, the present work will provide a novel clue to understand the regulatory mechanism for the kinetochore activity of CENP-N during the cell cycle.

Regulation of the Histone Deacetylase Hst3 by Cyclin-dependent Kinases and the Ubiquitin Ligase SCFCdc4

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is a modification of new H3 molecules deposited throughout the genome during S-phase. H3K56ac is removed by the sirtuins Hst3 and Hst4 at later stages of the cell cycle. Previous studies indicated that regulated degradation of Hst3 plays an important role in the genome-wide waves of H3K56 acetylation and deacetylation that occur during each cell cycle. However, little is known regarding the mechanism of cell cycle-regulated Hst3 degradation. Here, we demonstrate that Hst3 instability in vivo is dependent upon the ubiquitin ligase SCF(Cdc4) and that Hst3 is phosphorylated at two Cdk1 sites, threonine 380 and threonine 384. This creates a diphosphorylated degron that is necessary for Hst3 polyubiquitylation by SCF(Cdc4). Mutation of the Hst3 diphospho-degron does not completely stabilize Hst3 in vivo, but it nonetheless results in a significant fitness defect that is particularly severe in mutant cells treated with the alkylating agent methyl methanesulfonate. Unexpectedly, we show that Hst3 can be degraded between G2 and anaphase, a window of the cell cycle where Hst3 normally mediates genome-wide deacetylation of H3K56. Our results suggest an intricate coordination between Hst3 synthesis, genome-wide H3K56 deacetylation by Hst3, and cell cycle-regulated degradation of Hst3 by cyclin-dependent kinases and SCF(Cdc4).

Involvement of the single Cul4 gene of Chinese mitten crab Eriocheir sinensis in spermatogenesis

The Cullin-RING finger ligases (CRLs) are involved in the ubiquitin-mediated degradation of cell cycle regulators and play an important role in gametogenesis. Cullin 4 (CUL4) is a conserved core component of a new class of ubiquitin E3 ligase, and participates in the proteolysis of several regulatory proteins through the ubiquitin–proteasome pathway. The mammals encode two paralogs of CUL4, CUL4A and CUL4B, and the two Cul4 genes are functionally redundant. However, Drosophila or other metazoans only contain one Cul4 gene. Here we cloned the Cul4 gene and confirmed that there is only one protein of CUL4 in Eriocheir sinensis, a full length Cul4 comprised of 2777 nucleotides, an open-reading frame of 2373bp encoding 790 amino acid residues. The expression level of Cul4 mRNAs, as demonstrated by quantitative real-time PCR, varied significantly during testis development, with the greatest transcript levels found at an early stage. Localization analysis using antibodies against CUL4A/4B in the reproductive system showed that EsCUL4 mainly distribute in spermatogonia and primary spermatocytes, and gradually reduced during the development and maturation of sperm. The results indicated that a single CUL4 protein may play a role in spermatogenesis in E. sinensis.

Identification of She3 as an SCFGrr1 Substrate in Budding Yeast

The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF) complex. Acting in concert with the substrate-binding F-box protein Grr1, SCFGrr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth.

Loss-of-Function Screen Reveals Novel Regulators Required forDrosophilaGermline Stem Cell Self-Renewal

The germline stem cells (GSCs) of Drosophila melanogaster ovary provide an excellent model system to study the molecular mechanisms of stem cell self-renewal. To reveal novel factors required for Drosophila female GSC maintenance and/or division, we performed a loss-of-function screen in GSCs by using a collection of P-element–induced alleles of essential genes. Mutations in genes of various functional groups were identified to cause defects in GSC self-renewal. Here we report that a group of mutations affecting various ubiquitin-conjugating enzymes cause significant GSCs loss, including Plenty of SH3s (POSH), Ubiquitin-conjugating enzyme 10 (UbcD10), and pineapple eye (pie). Ubiquitin-mediated protein degradation plays a variety of roles in the regulation of many developmental processes, including mediating stem cell division through degradation of cell cycle regulators. We demonstrated that pie, sharing highly conserved RING domains with human E3 ubiquitin ligase G2E3 that are critical for early embryonic development, is specifically required for GSC maintenance, possibly through regulation of bone morphogenetic protein signaling pathway. Despite the previously reported role in imaginal disc cell survival, pie loss-of-function induced GSC loss is not to the result of caspase-involved cell death. Further efforts are needed to elucidate the functions of ubiquitin ligases in GSC maintenance, which will ultimately contribute to a better understanding of how the ubiquitin-conjugating enzymes regulate stem cell biology in mammalian systems.

Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control

The covalent modification of proteins with ubiquitin is required for accurate cell division in all eukaryotes. Ubiquitylation depends on an enzymatic cascade, in which E3 enzymes recruit specific substrates for modification. Among ~600 human E3s, the SCF (Skp1-cullin1-F-box) and the APC/C (anaphase-promoting complex/cyclosome) are known for driving the degradation of cell cycle regulators to accomplish irreversible cell cycle transitions. The cell cycle machinery reciprocally regulates the SCF and APC/C through various mechanisms, including the modification of these E3s or the binding of specific inhibitors. Recent studies have provided new insight into the intricate relationship between ubiquitylation and the cell division apparatus as they revealed roles for atypical ubiquitin chains, new mechanisms of substrate and E3 regulation, as well as extensive crosstalk between ubiquitylation enzymes. Here, we review these emerging regulatory mechanisms of ubiquitin-dependent cell cycle control and discuss how their manipulation might provide therapeutic benefits in the future.

Characterization of MRFAP1 turnover and interactions downstream of the NEDD8 pathway.

The NEDD8-Cullin E3 ligase pathway plays an important role in protein homeostasis, in particular the degradation of cell cycle regulators and transcriptional control networks. To characterize NEDD8-cullin target proteins, we performed a quantitative proteomic analysis of cells treated with MLN4924, a small molecule inhibitor of the NEDD8 conjugation pathway. MRFAP1 and its interaction partner, MORF4L1, were among the most up-regulated proteins after NEDD8 inhibition in multiple human cell lines. We show that MRFAP1 has a fast turnover rate in the absence of MLN4924 and is degraded via the ubiquitin-proteasome system. The increased abundance of MRFAP1 after MLN4924 treatment results from a decreased rate of degradation. Characterization of the binding partners of both MRFAP1 and MORF4L1 revealed a complex protein-protein interaction network. MRFAP1 bound to a number of E3 ubiquitin ligases, including CUL4B, but not to components of the NuA4 complex, including MRGBP, which bound to MORF4L1. These data indicate that MRFAP1 may regulate the ability of MORF4L1 to interact with chromatin-modifying enzymes by binding to MORF4L1 in a mutually exclusive manner with MRGBP. Analysis of MRFAP1 expression in human tissues by immunostaining with a MRFAP1-specific antibody revealed that it was detectable in only a small number of tissues, in particular testis and brain. Strikingly, analysis of the seminiferous tubules of the testis showed the highest nuclear staining in the spermatogonia and much weaker staining in the spermatocytes and spermatids. MRGBP was inversely correlated with MRFAP1 expression in these cell types, consistent with an exchange of MORF4L1 interaction partners as cells progress through meiosis in the testis. These data highlight an important new arm of the NEDD8-cullin pathway.

Non‐proteolytic ubiquitylation counteracts the APC/C‐inhibitory function of XErp1

Mature Xenopus oocytes are arrested in meiosis by the activity of XErp1/Emi2, an inhibitor of the ubiquitin-ligase anaphase-promoting complex/cyclosome (APC/C). On fertilization, XErp1 is degraded, resulting in APC/C activation and the consequent degradation of cell-cycle regulators and exit from meiosis. In this study, we show that a modest increase in the activity of the ubiquitin-conjugating enzyme UbcX overrides the meiotic arrest in an APC/C-dependent reaction. Intriguingly, XErp1 remains stable in these conditions. We found that UbcX causes the ubiquitylation of XErp1, followed by its dissociation from the APC/C. Our data support the idea that ubiquitylation regulates the APC/C-inhibitory activity of XErp1.

Anaphase-Promoting Complex/Cyclosome Participates in the Acute Response to Protein-Damaging Stress

The ubiquitin E3 ligase anaphase-promoting complex/cyclosome (APC/C) drives degradation of cell cycle regulators in cycling cells by associating with the coactivators Cdc20 and Cdh1. Although a plethora of APC/C substrates have been identified, only a few transcriptional regulators are described as direct targets of APC/C-dependent ubiquitination. Here we show that APC/C, through substrate recognition by both Cdc20 and Cdh1, mediates ubiquitination and degradation of heat shock factor 2 (HSF2), a transcription factor that binds to the Hsp70 promoter. The interaction between HSF2 and the APC/C subunit Cdc27 and coactivator Cdc20 is enhanced by moderate heat stress, and the degradation of HSF2 is induced during the acute phase of the heat shock response, leading to clearance of HSF2 from the Hsp70 promoter. Remarkably, Cdc20 and the proteasome 20S core α2 subunit are recruited to the Hsp70 promoter in a heat shock-inducible manner. Moreover, the heat shock-induced expression of Hsp70 is increased when Cdc20 is silenced by a specific small interfering RNA (siRNA). Our results provide the first evidence for participation of APC/C in the acute response to protein-damaging stress.

The degradation of cell cycle regulators by SKP2/CKS1 ubiquitin ligase is genetically controlled in rodent liver cancer and contributes to determine the susceptibility to the disease

Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21(WAF1), p27(KIP1), p57(KIP2) and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21(WAF1), p27(KIP1), p57(KIP), p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1-Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21(WAF1), P27(KIP1), P57(KIP2) and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators.

SKP2 and CKS1 Promote Degradation of Cell Cycle Regulators and Are Associated With Hepatocellular Carcinoma Prognosis

The cell cycle regulators P21(WAF1), P27(KIP1), P57(KIP2), P130, RASSF1A, and FOXO1 are down-regulated during hepatocellular carcinoma (HCC) pathogenesis. We investigated the role of the ubiquitin ligase subunits CKS1 and SKP2, which regulate proteasome degradation of cell cycle regulators, in HCC progression. Human HCC tissues from patients with better (HCCB, >3 years survival) and poorer prognosis (HCCP, <3 years survival) and HCC cell lines were analyzed. The promoters of P21(WAF1), P27(KIP1), and P57(KIP2) were more frequently hypermethylated in HCCP than HCCB. Messenger RNA levels of these genes were up-regulated in samples in which these genes were not methylated; protein levels increased only in HCCB because of CKS1- and SKP2-dependent ubiquitination of these proteins in HCCP. The level of SKP2 expression correlated with rate of HCC cell proliferation and level of microvascularization of samples and was inversely correlated with apoptosis and survival. In HCCB, SKP2 activity was balanced by degradation by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-CDH1 and up-regulation of SKP2 suppressor histidine triad nucleotide binding protein 1 (HINT1). In HCCP, however, SKP2 was not degraded because of down-regulation of the phosphatase CDC14B, CDK2-dependent serine phosphorylation (which inhibits interaction between CDH1 and SKP2), and HINT1 inactivation. In HCC cells, small interfering RNA knockdown of SKP2 reduced proliferation and ubiquitination of the cell cycle regulators, whereas SKP2 increased proliferation and reduced expression of cell cycle regulators. Ubiquitination and proteasome degradation of P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A, and FOXO1 and mechanisms that prevent degradation of SKP2 by APC/C-CDH1 contribute to HCC progression. CKS1-SKP2 ligase might be developed as a therapeutic target or diagnostic marker.

Nucleus-Specific and Cell Cycle-Regulated Degradation of Mitogen-Activated Protein Kinase Scaffold Protein Ste5 Contributes to the Control of Signaling Competence

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G(1) phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF(Cdc4) ubiquitin ligase and requires phosphorylation by the G(1) cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G(1) Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

Cell Cycle Control by the CDC25 Phosphatases

Cell division cycle 25 (CDC25) phosphatases are key actors in eukaryotic cell cycle control. They are responsible for the dephosphorylations that activate the cyclin-dependent kinases (CDK) at specific stages of the cell cycle. Human CDC25A, CDC25B and CDC25C are also central targets and regulators of the G2/M checkpoint mechanisms activated in response to DNA injury. The expression and activity of these enzymes is finely regulated by multiple mechanisms including post-translational modifications, interactions with regulatory partners, control of their intracellular localization, and cell cycle-regulated degradation. Altered expression of these phosphatases is associated with checkpoint bypass and genetic instability. Accordingly, increased expression of CDC25A and CDC25B is found in many high-grade tumors and is correlated with poor prognosis in human cancers. This review summarizes our current knowledge within this domain and discusses the data that support therapeutic strategies targeting CDC25 activity in the treatment of cancer.

Unscheduled Sister-Chromatid-Separation in the Presence of Spindle Disruption in Acute Myelogenous Leukemia

Mitosis is known to be one of the most critical events in the cell cycle. The spindle assembly checkpoint (SAC) is required for proper chromosome segregation during mitosis. The SAC serves as a mitotic surveillance mechanism responsible for detection of misassembly of chromosomes to the mitotic spindle. Lack of chromosome attachment and spindle tension generate a specific „wait-anaphase-signal“. This particular signal interferes with proteolysis, depending on the ubiquitin-ligase APCCdc20, thereby inhibiting mitotic progression through stabilization of mitotic regulators. We found several AML cell lines to be incapable of properly accumulating in mitosis upon nocodazole-induced spindle disruption when compared to a set of Burkitt's lymphoma cell lines. This result was further supported by the degradation of the mitotic regulators Cyclin B and Securin in synchronized Kasumi-1 cells in the presence of nocodazole shortly after entering mitosis. Interestingly, the SAC proteins BubR1 and Bub1 were found at low expression levels in AML cell lines in comparison to Burkitt's lymphoma cell lines. We established a shRNA-based model to evaluate the effects of BubR1- and/or Bub1-repression to levels found in AML cell lines to directly compare the Bub1/BubR1 knockdown phenotype with the investigated AML cell lines. Our findings support the view that BubR1 repression alone is sufficient to confer SAC deficiency. To determine the frequency of BubR1 repression in patient-derived primary cells, AML blasts were cytokine-stimulated to enter the cell cycle. Flow cytometry-based G2/M-specific expression analysis of BubR1 in primary AML blasts revealed lower expression in most analyzed cell populations. To further test the hypothesis that AML cells override the metaphase-to-anaphase-transition despite spindle damage, we performed Giemsa staining of cells that were incubated in nocodazole containing growth medium. In AML cell lines, unlike the analyzed Burkitt's lymphoma cell lines, a significant number of metaphase-like cells contained single chromatids, suggesting premature sister chromatid separation in the presence of spindle damage. Premature sister-chromatid-separation in the presence of chromosomal misalignment would lead to aneuploidy and favor the onset of genomic instability. Our recent efforts focus on high-throughput automated live cell scanning, promising a better understanding of cell division and chromosome separation in the context of different challenges, such as spindle damage. This powerful tool allows a more precise characterization of our knockdown phenotypes in the double-knockdown system, which is a prerequisite for comparison of our model system with AML cell lines. Finally, this new technique might also prove useful to extend our analyses to patient derived AML blasts. As we observed deregulation of SAC protein levels in AML cell lines and primary AML blasts, our findings of premature degradation of cell cycle regulators and unscheduled sister-chromatid-separation suggest an important role for SAC malfunction in the development of AML with karyotypic abnormalities. Mitotic kinases, such as Plk1 and Aurora, are already promising targets for modern antineoplastic therapies. A deeper understanding of mitotic control in AML might contribute to even more sophisticated targeted therapeutic approaches.

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

Cyclin-dependent Kinases Phosphorylate Human Cdt1 and Induce Its Degradation

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.

Regulation of the ubiquitin proteasome pathway in human lens epithelial cells during the cell cycle

Most proliferating cells follow a series of orderly transitions from one phase to another. These transitions are usually controlled by timed degradation of cell cycle regulators by the ubiquitin-proteasome pathway (UPP). There are no published reports regarding the timing of phases of the human lens cell cycle or regarding cell cycle-related changes in UPP components. Objectives of this study were to characterize the timing of the phases of the human lens epithelial cell cycle and to explore potential functions of critical components of the UPP in controlling lens cell cycle. Human lens epithelial cells were synchronized at G0/G1 phase by contact inhibition. Cell cycle progression upon subculturing was monitored by FACS analysis. It took ∼40 hr for HLEC to complete one cell cycle, ∼20 hr for G1 phase, ∼8–10 hr for S phase and ∼10 hr for the combination of G2 and M phases. Proteasome-dependent degradation of p21 WAF and p27 Kip, the dominant Cdk inhibitors, was associated with the G1/S phase transition in these cells. Proteasome inhibition experiments indicate that proteolysis is the predominant process which is responsible for the variations in these regulators during the cell cycle. Levels of specific ubiquitin conjugating enzymes, Ubc7 and Ubc10, increased 6 and 2-fold at the G2/M phase and S/G2/M phases, respectively. Levels of these E2s decreased precipitously upon completion of the M phase. In contrast, levels of ubiquitin activating enzyme (E1) and Ubc3 remained constant during the cell cycle. Cul1, a component of the SCF (an E3), remained relatively constant during cell cycle. The up-regulation of Ubc7 and Ubc10 during the G2/M and S/G2/M phases suggests that these enzymes may be involved in controlling the cell cycle progression at this phase. Taken together, the data indicate that expression of key components of the UPP in the human lens epithelial cells is regulated in a cell cycle-dependent manner. Some of the variations in levels of ubiquitin conjugating enzymes are suggestive of previously undescribed functions.