Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.Endothelial cell (EC) senescence is accompanied with vascular dysfunction, including arterial stiffening and remodeling,¹ impaired angiogenesis,^{2, 3} reduced endothelial repair capability and increased incidence of cardiovascular disease.^{4, 5, 6} Cellular senescence can occur in vivo or in vitro in response to various stressors,^{7, 8, 9, 10} leading to suppression of cell proliferation. EC senescence has been reported to contribute to the pathogenesis of age-associated vascular diseases, such as atherosclerosis.¹¹ Thus, further understanding the mechanisms of EC senescence may help to identify effective targets for antisenescence therapy and treatment aging-associated cardiovascular disorders.Previous studies have shown that the secreted matricellular protein thrombospondin-1 (TSP1) is as potent inhibitor of angiogenesis¹² and its antiangiogenic activity is mediated by its receptors, CD36^{13, 14} and CD47.^{15, 16} CD47 is a ubiquitously expressed transmembrane protein that serves as a ligand for signal regulatory protein-α and is a signaling receptor of TSP1. The TSP1-CD47 pathway has an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation and atherosclerotic response.¹⁷ Ligation of CD47 by TSP1 has been shown to inhibit nitric oxide (NO)/cGMP signaling in vascular cells, leading to suppression of angiogenic responses.¹⁶ Recently, it was reported that lack of CD47 expression in ECs may enable these cells to spontaneously gain characteristics of embryonic stem cells.¹⁸ However, the potential role of CD47 in regulation of EC senescence has not been well explored. The present study was initiated to determine the role and mechanisms of TSP1-CD47 signaling pathway in regulating cell cycle progression and replicative senescence of ECs.     

The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

FZR2/CCS52A1 Expression Is a Determinant of Endoreduplication and Cell Expansion in Arabidopsis

Endoreduplication, a modified cell cycle that allows cells to increase ploidy without subsequent cell division, is a key component of plant growth and development. In this work, we show that some, but not all, of the endoreduplication of Arabidopsis (Arabidopsis thaliana) is mediated by the expression of a WD40 gene, FIZZY-RELATED2 (FZR2). Loss-of-function alleles show reduced endoreduplication and reduced expansion in trichomes and other leaf cells. Misexpression of FZR2 is sufficient to drive ectopic or extra endoreduplication in leaves, roots, and flowers, leading to alteration of cell sizes and, sometimes, organ size and shape. Our data, which suggest that reduced cell size can be compensated by increased cell proliferation to allow normal leaf morphology, are discussed with respect to the so-called compensation mechanism of plant development.A key element of plant morphogenesis is the balance among cell proliferation, expansion, and differentiation to produce organs of the characteristic sizes and shapes. At the heart of plant morphogenesis is the cell versus organism debate. On one hand, the cell theory postulates that the cells of a multicellular organism behave autonomously, and the sum of their activities results in the morphology of that organism (Schwann, 1839). On the other hand, the organismal theory postulates that morphology is governed by a genetic mechanism separate from the behavior of individual cells (Kaplan and Hagemann, 1991). Regardless of perspective, it is clear that the plant program of cell growth and proliferation is dynamic and responsive, i.e. perturbations in one factor can be compensated by modifications in the other (Day and Lawrence, 2000; Mizukami, 2001). For example, forcing a decrease in leaf cell number by overexpressing the cell cycle regulator KRP2 results in an increase in cell volume (De Veylder et al., 2001). Conversely, an increase in cell volume caused by the overexpression of ABP1 leads to a decrease in cell number (Jones et al., 1998). This compensation mechanism has been incorporated into a hybrid cell/organismal theory called “neo-cell theory” (Tsukaya, 2003). The nature of this compensation mechanism is unclear, although it likely involves cellular reaction to organ-level positional information.During the development of many plants, certain cell types undergo extensive endoreduplication, a modified cell cycle that results in DNA replication without subsequent mitosis. In leaves of Arabidopsis (Arabidopsis thaliana), several rounds of endoreduplication occur in trichomes and in most epidermal pavement cells, while guard cells remain diploid (Melaragno et al., 1993). In the last decade, extensive research has looked at the trichome as a model to unravel the mechanism controlling endoreduplication. During trichome development, four rounds of endoreduplication regularly take place, although variation exists (Melaragno et al., 1993). Some Arabidopsis mutants show concomitant changes in trichome ploidy and branch number, supporting a role of endoreduplication in the control of branch number (Hülskamp et al., 1994; Perazza et al., 1999; Downes et al., 2003). Branching changes are not always tied to endoploidy changes, however; for example, plants with loss-of-function alleles or overexpression of STICHEL show trichome branch number alterations without changes in nuclear DNA content (Ilgenfritz et al., 2003). Endoreduplication appears to be an important determinant of cell and organ size. Indeed, a correlation between cell size and endoploidy has been found in Arabidopsis and other plant species (Galbraith et al., 1991; Traas et al., 1998; Kudo and Kimura, 2002), although Arabidopsis root cells constitute an exception (Beemster et al., 2002), and certain genetic manipulations can also uncouple this relationship (Hemerly et al., 1993; Wang, et al., 2000; De Veylder et al., 2001; Fujikura et al., 2007). Interestingly, different levels of endoreduplication may affect the same tissue in distinct species. For instance, orchid (Oncidium varicosum and Phalaenopsis spp.) and cabbage (Brassica capitata) petals are large and undergo extensive endoreduplication, with some cells reaching 64C (Kudo and Kimura, 2001; Lee et al., 2004). The sizes of petal cells, and hence of petal organs, directly correlate with their ploidy in these studies. By contrast, the cone cells of Arabidopsis petals remain diploid despite being capable of endoreduplication (Hase et al., 2005), and this plant shows relatively small flowers in comparison to close relatives. Unlike orchid and cabbage, Arabidopsis is self-fertilizing, so producing small petals would not hinder its reproduction.Similar cellular components regulate endoreduplication and the mitotic cell cycle. Cyclin-dependent kinases (CDKs) control the cell cycle by forming complexes with cyclins, by phosphorylation or dephosphorylation, or by association with CDK inhibitors. Expression and degradation of specific cyclins promote progression through distinct stages of the cell cycle and control the exit from the cell cycle. For example, D-type cyclins become phosphorylated and associate with the A-type CDK to induce the switch from G1 phase to S phase (Inzé and De Veylder, 2006). Destruction of B1-type cyclins seems to be necessary for completion of the M phase, because introduction of a B1 cyclin for which the destruction-box motif has been altered can prevent cytokinesis (Weingartner et al., 2004). In yeast and Drosophila, Fizzy-Related (FZR) family proteins have been shown to trigger the degradation of A- and B-type cyclins by targeting them to the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin protein ligase (Sigrist and Lehner, 1997; Yamaguchi et al., 1997). In those studies, misexpression of FZR family proteins was able to induce endoreduplication. Furthermore, a recent study linked the APC/C to local cell expansion, endoreduplication, and the compensation mechanism in Arabidopsis (Serralbo et al., 2006).The plant homologs of FZR were first implicated in endoreduplication in root nodules of Medicago sativa, and antisense expression of the Medicago truncatula Cell-cycle Switch 52 A and B (Ccs52A and Ccs52B), orthologs of FZR, led to reduced endoreduplication in that species (Cebolla et al., 1999). In Arabidopsis, three FZR homologs exist, and in cell cultures, FZR1 and FZR2 showed similar high expression in G1 and S phases of the cell cycle, while FZR3 expression increased at the end of G2 and beginning of M. All three FZR proteins associated to free and CDK-bound A- and B-type cyclins (Fülöp et al., 2005). Although the FZR genes are known to be expressed in all major tissues (Beemster et al., 2005), the normal developmental function of the FZR family has not yet been determined in Arabidopsis.In this study, we examined the developmental roles of one of the Arabidopsis FZR homologs, FZR2. We found that FZR2 expression is necessary for correct cell expansion and endoreduplication, and its misexpression is sufficient to induce extra or ectopic endoreduplication and cell expansion. We gathered empirical data that address the putative compensation mechanism balancing cell proliferation with cell expansion.     

NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism

Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies.We, and others, have demonstrated that the outcome of CD40 receptor activation by its ligand CD40L/CD154 in carcinomas predominantly relies on the way the ligand is presented to the receptor at the cell surface, where membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) induces cell growth arrest and apoptosis. mCD40L-induced cell death occurs through a mechanism that involves the downregulation of pro-survival signals, mainly the PI3K/AKT pathway and the sustained activation of the pro-apoptotic JNK pathway, leading to caspase activation and subsequently apoptosis.^{1, 2} However, the role of CD40L in cell cycle regulation and growth arrest are still largely elusive; cell cycle regulation is a complex process that ensures correct cell division and is controlled by multiple mechanisms, governed by key regulatory proteins, including the cyclin-dependent kinases (CDKs) and their regulatory inhibitors. CDKs are a family of serine/threonine protein kinases that are activated at specific points of the cell cycle and are regulated by several different mechanisms.³CDK inhibitors function through inhibition of the G1 CDK cyclin complexes.⁴ Furthermore, the CDK inhibitor p21 has the ability to also inhibit DNA synthesis by binding to and inhibiting proliferating cell nuclear antigen.^{5, 6, 7} p21 protein expression is tightly regulated by the p53 tumour suppressor gene.⁸ The expression of p21 is also reported to be regulated by the recently identified Ras-associated factor 5 (NORE1A/RASSF5),⁹ a protein that is 41.8 kD and a member of the RASSF family of tumour suppressors.¹⁰ NORE1A expression is frequently downregulated by promoter methylation in human tumours.^{11, 12} Structurally, NORE1A contains a Ras-binding domain and was originally identified by a yeast two-hybrid screen.¹³ NORE1A can directly bind the Ras oncoprotein in a GTP-dependent manner.¹⁴ Exogenous expression of NORE1A has been reported to promote apoptosis by Ras-dependant and -independent mechanisms.¹² Furthermore, NORE1A protein expression results in a decrease in the number of cells in S phase of the cell cycle,¹⁵ indicating that NORE1A might function as a tumour suppressor; here, we show for the first time that mCD40L but not sCD40L induces the expression of NORE1A in an NFκB-dependant manner. NORE1A expression was found to contribute to mCD40L-induced cell death since inhibition of mCD40L-induced NORE1A expression resulted in reduced cell death by mCD40L. The role of CD40-induced NORE1A expression in cell cycle regulation was also examined and found to be mediated by p21 upregulation.     

Role of Retinoic Acid and Fibroblast Growth Factor 2 in Neural Differentiation from Cynomolgus Monkey (Macaca fascicularis) Embryonic Stem Cells

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.Abbreviations: EB, embryoid body; ES, embryonic stem; ESM, embryonic stem cell medium; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; MBP, myelin basic protein; RA, retinoic acid; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigenPluripotent stem cells are potential sources of material for cell replacement therapy and are useful experimental tools for in vitro models of human disease and drug screening. Embryonic stem (ES) cells are capable of extensive proliferation and multilineage differentiation, and thus ES-derived cells are suitable for use in cell-replacement therapies.^18,23 Reported ES cell characteristics including tumorigenic potential, DNA methylation status, expression of imprinted genes, and chromatin structure were elucidated by using induced pluripotent stem cells.^2,11,17 Because the social expectations of regeneration medicine are growing, we must perform basic research with ES cells, which differ from induced pluripotent stem cells in terms of origin, differentiation ability, and epigenetic status.^2,8Several advances in research have been made by using mouse ES cells. Furthermore, primate ES cell lines have been established from rhesus monkeys (Macaca mulatta),²⁴ common marmosets (Callithrix jacchus),²⁵ cynomolgus monkeys (M. fascicularis),²⁰ and African green monkeys (Chlorocebus aethiops).¹⁹ Mouse and other mammalian ES cells differ markedly in their responses to the signaling pathways that support self-renewal.^8,28 Mouse ES cells require leukemia inhibitory factor (LIF)–STAT3 signaling.¹⁴ In contrast, primate ES cells do not respond to LIF. Fibroblast growth factor 2 (FGF2) appears to be the most upstream self-renewal factor in primate ES cells. FGF2 also exerts its effects through indirect mechanisms, such as the TGFβ–Activin–Nodal signaling pathway, in primate ES cells.²¹ In addition to the biologic similarities between monkeys and humans, ES cells derived from cynomolgus monkeys or human blastocysts have extensive similarities that are not apparent in mouse ES cells.^8,14,21,28 Numerous monkey ES cell lines are now available, and cynomolgus monkeys are an efficient model for developing strategies to investigate the efficacy of ES-cell–based medical treatments in humans.Several growth factors and chemical compounds, including retinoic acid (RA),^4,9,13,22,26 FGF2,^9,10,16,22 epidermal growth factor,^9,22 SB431542,^1,4,10 dorsomorphin,^10,27 sonic hedgehog,^{12,13,16,27,29} and noggin,^1,4,9,27 are essential for the differentiation and proliferation or maintenance of neural stem cells derived from primate ES cells. Of these factors, active RA signaling suppresses a mesodermal fate by inhibiting Wnt and Nodal signaling pathways during in vitro culture and leads to neuroectoderm differentiation in ES cells.^4,13,26 RA is an indispensable factor for the specialization to neural cells. FGF2 is important during nervous system development,¹² and FGF2 and RA both are believed to influence the differentiation to neural cells. The current study was done to clarify the mechanism of RA and FGF2 in the induction of differentiation along the neural lineage.We recently established a monkey ES cell line that does not need FGF2 supplementation for maintenance of the undifferentiated state. This ES cell line allowed us to study the role of differentiation to neural cells with RA and enabled us to compare ES cell differentiation in the context of supplementation with RA or FGF2 in culture. To this end, we established a novel cynomolgus monkey cell line derived from ES cells and maintained it in an undifferentiated state in the absence of FGF2 supplementation.     

Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G₂/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G₂/M checkpoint-mediated apoptosis.Cell division cycle 25 (Cdc25) phosphatases are dual-specificity phosphatases involved in cell cycle regulation. By removing inhibitory phosphate groups from phospho-Thr and phospho-Tyr residues of cyclin-dependent kinases (CDKs),¹ Cdc25 proteins regulate cell cycle progression in S phase and mitosis. In mammals, three isoforms of Cdc25 phosphatases have been reported: Cdc25A, which controls the G₁/S transition;^{2, 3} Cdc25B, which is a mitotic starter;⁴ and Cdc25C, which controls the G₂/M phase.⁵ Overexpression of Cdc25 phosphatases is frequently associated with various cancers.⁶ Upon exposure to DNA-damaging reagents like UV radiation or free oxygen radicals, Cdc25 phosphatases are key targets of the checkpoint machinery, resulting in cell cycle arrest and apoptosis. The 14-3-3 proteins bind to phosphorylated Ser-216 of Cdc25C and induce Cdc25C export from the nucleus during interphase in response to DNA damage,^{7, 8} but they have no apparent effect on Cdc25C phosphatase activity.^{9, 10} In addition, hyperphosphorylation of Cdc25C correlates to its enhanced phosphatase activity.¹¹ Most studies with Cdc25C have focused on its role in mitotic progression. However, the role of Cdc25C is not clear when it is sequestered in the cytoplasm by binding to 14-3-3.Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAPKKK5), is a ubiquitously expressed enzyme with a molecular weight of 170 kDa. The kinase activity of ASK1 is stimulated by various cellular stresses, such as H₂O₂,^{12, 13} tumor necrosis factor-α (TNF-α),¹⁴ Fas ligand,¹⁵ serum withdrawal,¹³ and ER stress.¹⁶ Stimulated ASK1 phosphorylates and activates downstream MAP kinase kinases (MKKs) involved in c-Jun N-terminal kinase (JNK) and p38 pathways.^{17, 18, 19} Phosphorylation and activation of ASK1 can induce apoptosis, differentiation, or other cellular responses, depending on the cell type. ASK1 is regulated either positively or negatively depending on its binding proteins.^{12, 13, 15, 18, 19, 20, 21, 22, 23, 24, 25}ASK1 is regulated by phosphorylation at several Ser/Thr/Tyr residues. Phosphorylation at Thr-838 leads to activation of ASK1, whereas phosphorylation at Ser-83, Ser-967, or Ser-1034 inactivates ASK1.^{24, 26, 27, 28} ASK1 is basally phosphorylated at Ser-967 by an unidentified kinase, and 14-3-3 binds to this site to inhibit ASK1.²⁴ Phosphorylation at Ser-83 is known to be catalyzed by Akt or PIM1.^{27, 29} Oligomerization-dependent autophosphorylation at Thr-838, which is located in the activation loop of the kinase domain, is essential for ASK1 activation.^{14, 18, 30} Phosphorylation at Tyr-718 by JAK2 induces ASK1 degradation.³¹ Several phosphatases that dephosphorylate some of these sites have been identified. Serine/threonine protein phosphatase type 5 (PP5) and PP2C dephosphorylate phosphorylated (p)-Thr-838,^{28, 32} whereas PP2A and SHP2 dephosphorylate p-Ser-967 and p-Tyr-718, respectively.^{31, 33} Little is known about the kinase or phosphatase that regulates phosphorylation at Ser-1034. Although ASK1 phosphorylation is known to be involved in the regulation of apoptosis, only a few reports show that ASK1 phosphorylation or activity is dependent on the cell cycle.^{21, 34}In this study, we examined the functional relationship between Cdc25C and ASK1 and identified a novel function of Cdc25C phosphatase that can dephosphorylate and inhibit ASK1 in interphase but not in mitosis. Furthermore, we demonstrated that Cdc25C phosphorylation status plays a critical role in the interaction with and the activity of ASK1. These results reveal a novel regulatory function of Cdc25C in the ASK1-mediated apoptosis signaling pathway.     

Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development

Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAF^V600E melanomas.The cell cycle is a set of coordinated events that culminate in the formation of two cells from one mother cell. It''s composed of four major phases; G1 (growth phase 1), S (DNA synthesis phase), G2 (growth phase 2) and M (mitosis), which function to integrate environment sensing signaling pathways with cell growth and proliferation.¹ Cancer cells often deregulate the cell cycle and undergo unscheduled cell divisions, therefore inhibition of the cell cycle represents an opportunity for therapeutic intervention in treating proliferative diseases like cancer.² Most anti-cancer drugs perturb the proliferation cycle of tumor cells by inhibiting/damaging cell cycle events, which activate checkpoints, arrest cells and induce apoptosis.³ For example, inhibitors targeting DNA replication (5-fluorouracil) and cell division (microtubule-stabilizing paclitaxel) have been used successfully for treating a broad array of cancers including breast and colorectal cancers.² Nevertheless, due to toxicity issues, drugs targeting the cell division machinery like mitotic kinases (AurKA/B and Plk1) and kinesins (Kif11 and CENP-E) have been developed.³ However, these drugs have shown limited efficacy in vivo.⁴ Thus, there is a critical need to identify novel drug-like molecules that inhibit cancer cell cycle progression, which can be developed into novel cancer therapies.Genome wide studies aimed at depleting the expression of human genes and characterizing their contribution to cell cycle progression have generated a wealth of information regarding the enzymatic machinery required for proliferation.⁵ These enzymes have become the focus of targeted screening campaigns aimed at finding inhibitors to their activities. For example, an in vitro chemical screen targeting Plk1 identified the small molecule BI2536.⁶ BI2536 was not only used to define novel roles for Plk1 during cell division, it was further developed into an anti-cancer drug whose efficacy is being evaluated in clinical trials.⁷ Therefore, beyond their therapeutic potential, inhibitors can be used as molecular probes for dissecting the function of enzymes critical for cell cycle progression in an acute and temporal manner. However, there are no inhibitors to the majority of the cell cycle machinery and the discovery and characterization of such inhibitors would aid our ability to understand the mechanisms regulating cell division.Although molecularly targeted screens have grown in popularity, they rely on the previous identification and validation of specific cancer targets with druggable activities/interactions.⁸ As an alternative, unbiased high-throughput chemical screens have tried to identify inhibitors to a single cell cycle phase,^{9, 10, 11, 12, 13, 14, 15} which limited their ability to identify novel anti-proliferative agents to other phases of the cell cycle. Nonetheless, G2-phase, M-phase, and cytokinesis screens successfully identified inhibitors to Kif11, Plk1, RhoA, and microtubules.^{9, 10, 11, 12, 13, 14, 15} These inhibitors aided the functional characterization of these proteins and were instrumental for developing drugs with therapeutic potential. However, these screens were conducted with a limited number of compounds (100–38 000) or cell extract fractions, with several screens using the same library of 16 320 compounds, thus limiting compound diversity, chemical coverage, and opportunities for novel discoveries. Most screens also lacked chemical analyses to understand the physiochemical properties of bioactive compounds and their cellular targets. In addition, previous screens have not analyzed the four phases of the cell cycle as a biological system. Thus, there is a critical need to develop new screening strategies to discover novel anti-cancer drugs.This, prompted us to establish an integrated high-throughput screening cell-based strategy for identifying small molecule cell cycle modulators, for use in dissecting the mechanisms of cancer cell division, and for developing novel cancer therapies. We report the development of this novel cell-based screening platform, the discovery of cell cycle phase specific inhibitors, the chemical analyses of these inhibitors, the cell culture characterization of cell division inhibitors, and the detailed examination of MI-181, which has potent anti-cancer activity, especially against melanomas.