G1 Phase Progression Research Articles

Clinical significance of cell cycle inhibitors in hepatocellular carcinoma

It is well accepted that cell cycle regulators are strongly implicated in the progression of cancer development. p16 and p27 are potent cyclin-dependent kinase (CDK) inhibitors involved in G1 phase progression, and are regarded as adverse prognostic biomarkers for various types of cancers. It has been reported that the main mechanism for p16 inactivation is aberrant DNA methylation, while p27 is exclusively inactivated by proteasome-mediated protein degradation. We have found that p27 is decreased in around half of hepatocellular carcinomas (HCCs), and in some cases p27 is inactivated by inappropriate interaction with cyclin D1/CDK4 complexes. In such cases, p16 is concomitantly inactivated through DNA methylation. Taking into consideration the complex interaction between p16 and p27, a comprehensive analysis including p16 and p27 would be useful for predicting the prognosis of HCC patients.

Ssrp1a controls organogenesis by promoting cell cycle progression and RNA synthesis

Tightly controlled DNA replication and RNA transcription are essential for differentiation and tissue growth in multicellular organisms. Histone chaperones, including the FACT (facilitates chromatin transcription) complex, are central for these processes and act by mediating DNA access through nucleosome reorganisation. However, their roles in vertebrate organogenesis are poorly understood. Here, we report the identification of zebrafish mutants for the gene encoding Structure specific recognition protein 1a (Ssrp1a), which, together with Spt16, forms the FACT heterodimer. Focussing on the liver and eye, we show that zygotic Ssrp1a is essential for proliferation and differentiation during organogenesis. Specifically, gene expression indicative of progressive organ differentiation is disrupted and RNA transcription is globally reduced. Ssrp1a-deficient embryos exhibit DNA synthesis defects and prolonged S phase, uncovering a role distinct from that of Spt16, which promotes G1 phase progression. Gene deletion/replacement experiments in Drosophila show that Ssrp1b, Ssrp1a and N-terminal Ssrp1a, equivalent to the yeast homologue Pob3, can substitute Drosophila Ssrp function. These data suggest that (1) Ssrp1b does not compensate for Ssrp1a loss in the zebrafish embryo, probably owing to insufficient expression levels, and (2) despite fundamental structural differences, the mechanisms mediating DNA accessibility by FACT are conserved between yeast and metazoans. We propose that the essential functions of Ssrp1a in DNA replication and gene transcription, together with its dynamic spatiotemporal expression, ensure organ-specific differentiation and proportional growth, which are crucial for the forming embryo.

CDC25A targeting by miR-483-3p decreases CCND–CDK4/6 assembly and contributes to cell cycle arrest

Disruption of contact inhibition and serum afflux that occur after a tissue injury activate cell cycle, which then stops when confluence is reached again. Although the events involved in cell cycle entry have been widely documented, those managing cell cycle exit have remained so far ill defined. We have identified that the final stage of wound closure is preceded in keratinocytes by a strong accumulation of miR-483-3p, which acts as a mandatory signal triggering cell cycle arrest when confluence is reached. Blocking miR-483-3p accumulation strongly delays cell cycle exit, maintains cells into a proliferative state and retards their differentiation program. Using two models of cell cycle synchronization (i.e. mechanical injury and serum addition), we show that an ectopic upregulation of miR-483-3p blocks cell cycle progression in early G1 phase. This arrest results from a direct targeting of the CDC25A phosphatase by miR-483-3p, which can be impeded using an anti-miRNA against miR-483-3p or a protector that blocks the complex formation between miR-483-3p and the 3'-untranslated region (UTR) of CDC25A transcript. We show that the miRNA-induced silencing of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases which, in turn, abolishes CDK4/6 capacity to associate with D-type cyclins. This prevents CDK4/6 kinases' activation, impairs downstream events such as cyclin E stimulation and sequesters cells in early G1. We propose this new regulatory process of cyclin-CDK association as a general mechanism coupling miRNA-mediated CDC25A invalidation to CDK post-transcriptional modifications and cell cycle control.

Magnetic Shielding Accelerates the Proliferation of Human Neuroblastoma Cell by Promoting G1-Phase Progression

Organisms have been exposed to the geomagnetic field (GMF) throughout evolutionary history. Exposure to the hypomagnetic field (HMF) by deep magnetic shielding has recently been suggested to have a negative effect on the structure and function of the central nervous system, particularly during early development. Although changes in cell growth and differentiation have been observed in the HMF, the effects of the HMF on cell cycle progression still remain unclear. Here we show that continuous HMF exposure significantly increases the proliferation of human neuroblastoma (SH-SY5Y) cells. The acceleration of proliferation results from a forward shift of the cell cycle in G1-phase. The G2/M-phase progression is not affected in the HMF. Our data is the first to demonstrate that the HMF can stimulate the proliferation of SH-SY5Y cells by promoting cell cycle progression in the G1-phase. This provides a novel way to study the mechanism of cells in response to changes of environmental magnetic field including the GMF.

Involvement of Rho proteins in G1 phase cell cycle progression

Rho proteins, including RhoA, Rac1 and Cdc42, are members of the Ras superfamily of monomeric GTP-binding proteins, which are well-known regulators of the cytoskeleton. Numerous studies have shown that an intact cytoskeleton is required for cell cycle progression through G1 phase as well as mitosis and cytokinesis. Because of their role in both cytoskeletal rearrangement and mitogenic signaling, Rho family proteins are key mediators of cell cycle progression. In this paper, we review the current state of knowledge concerning the Rho-dependent signaling pathways that regulate the expression of cell cycle regulatory proteins required for G1 phase progression and S phase entry.

Overexpression of DOC-1R inhibits cell cycle G1/S transition by repressing CDK2 expression and activation.

DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.

Reciprocal Control of G1-Phase Progression Is Required for Th-POK/Runx3–Mediated CD4/8 Thymocyte Cell Fate Decision

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G(1) cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G(1)-phase extension and represses CD8 expression in a G(1) extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G(1) extension and CD8 repression. Importantly, forced G(1) extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G(1) progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G(1)-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

The Chromosome 9p21.3 Coronary Heart Disease Risk Allele Is Associated with Altered Gene Expression in Normal Heart and Vascular Tissues

Genome-wide association studies have identified a coronary artery disease (CAD) risk locus in a non-coding region at 9p21.3, the nearest genes being CDKN2A and CDKN2B. To understand the pathways by which this locus might influence CAD susceptibility, we investigated associations between the 9p21.3 risk genotype and global gene expression in heart tissue from donors with no diagnosed heart disease (n = 108, predominant cause of death, cerebral vascular accident) and in carotid plaque (n = 106), aorta (n = 104) and mammary artery (n = 88) tissues from heart valve and carotid endarterectomy patients. Genotyping was performed with Taqman assays and Illumina arrays, and gene expression profiles generated with Affymetrix microarrays. Associations were analyzed with an additive genetic model. In heart tissue, 46 genes were putatively altered in association with the 9p21.3 risk allele (70% down-regulated, fold-change >1.1 per allele, p<0.05 adjusted for age, gender, ethnicity, cause of death). These genes were enriched for biomarkers of myocardial infarction (p = 1.53×10−9), response to wounding (p = 2.65×10−10) and inflammatory processes (p<1.97×10−7). Among the top 10 most down-regulated genes, 7 genes shared a set of transcription factor binding sites within conserved promoter regions (p<1.14×10−5), suggesting they may be co-regulated. Canonical pathway modelling of the most differentially expressed transcripts across all tissues (154 genes, 60% down-regulated, fold-change >1.1 per allele, p<0.01) showed that 75% of the genes could be transcriptionally regulated through the cell cycle G1 phase progression pathway (p<1.08×10−258), in which CDKN2A and CDKN2B play a regulatory role. These data suggest that the cell cycle G1 phase progression pathway is activated in individuals with the 9p21.3 risk allele. This may contribute to a proliferative phenotype that promotes adverse cardiac hypertrophy and vascular remodeling, leading to an increased CAD risk.

Selenium promotes proliferation of chondrogenic cell ATDC5 by increment of intracellular ATP content under serum deprivation

Selenium (Se) is an essential micronutrient, and low Se intake in Se-deficient areas plays roles in an endemic osteochondropathy characterized by chondronecrosis in growth plate and articular cartilage. However, the biological activities of Se on cartilage are largely unknown. In this study, we examined the effects of Se on chondrogenic cell ATDC5 and the possible mechanisms involved. We demonstrated that Se stimulated ATDC5 cell proliferation under serum deprivation but not routine culture. Furthermore, Se promoted G1-phase cell cycle progression along with induction of cyclin D1 expression at the mRNA and protein level. Moreover, Se increased intracellular ATP content and decreased intracellular superoxide anion concentration without affecting intracellular redox status as estimated by ratio of the reduced and oxidized glutathione. In addition, suppression of intracellular ATP synthesis by glycolysis inhibitor or mitochondrial uncoupler both abrogated Se-mediated cyclin D1 induction. These findings suggest Se stimulates proliferation of chondrogenic cell ATDC5 through acceleration of cell cycle progression accompanied with cyclin D1 induction by enhancement of intracellular ATP content. This novel finding provides evidence for a role of Se in cartilage formation and degenerative processes and further supports the relationship between Se status and cartilage function that may lead to better utilization of Se for cartilage homeostasis.

Development of Ultra-Super Sensitive Immunohistochemistry and Its Application to the Etiological Study of Adult T-Cell Leukemia/Lymphoma

Antigen retrieval (AR) and ultra-super sensitive immunohistochemistry (ultra-IHC) have been established for application to archival human pathology specimens. The original ultra-IHC was the ImmunoMax method or the catalyzed signal amplification system (ImmunoMax/CSA method), comprising the streptavidin-biotin complex (sABC) method and catalyzed reporter deposition (CARD) reaction with visualization of its deposition. By introducing procedures to diminish non-specific staining in the original ultra-IHC method, we developed the modified ImmunoMax/CSA method with AR heating sections in an AR solution (heating-AR). The heating-AR and modified ImmunoMax/CSA method visualized expression of the predominantly simple present form of HTLV-1 proviral DNA pX region p40Tax protein (Tax) in adult T-cell leukemia/lymphoma (ATLL) cells in archival pathology specimens in approximately 75% of cases. The simple present form of Tax detected exhibited a close relation with ATLL cell proliferation. We also established a new simplified CSA (nsCSA) system by replacing the sABC method with the secondary antibody- and horse radish peroxidase-labeled polymer reagent method, introducing the pretreatments blocking non-specific binding of secondary antibody reagent, and diminishing the diffusion of deposition in the CARD reaction. Combined with AR treating sections with proteinase K solution (enzymatic-AR), the nsCSA system visualized granular immunostaining of the complex present form of Tax in a small number of ATLL cells in most cases, presenting the possibility of etiological pathological diagnosis of ATLL and suggesting that the complex present form of Tax-positive ATLL cells were young cells derived from ATLL stem cells. The heating-AR and ultra-IHC detected physiological expression of the p53 protein and its probable phosphorylation by Tax in peripheral blood mononuclear cells of peripheral blood tissue specimens from HTLV-1 carriers, as well as physiological and pathological expression of the molecules involved with G1 phase progression and G1–S phase transition (E2F-1, E2F-4, DP-1, and cyclin E) in ATLL and peripheral T-cell lymphoma cells. The ultra-IHC with AR is useful for etiological pathological diagnosis of ATLL since HTLV-1 pathogenicity depends on that of Tax, and can be a useful tool for studies translating advanced molecular biology and pathology to human pathology.

Eight paths of ERK1/2 signalling pathway regulating hepatocyte proliferation in rat liver regeneration

Although it is known that hormones, growth factors and integrin promote hepatocyte proliferation in liver regeneration (LR) through ERK1/2 signalling pathway, reports about regulating processes of its intracellular paths in hepatocytes of LR are limited. This study aims at exploring which paths of ERK1/2 signalling pathway participate in the regulation of rat LR, especially in hepatocyte proliferation, and how they do so. In all, 14 paths and 165 genes are known to be involved in ERK1/2 signalling pathway. Of them, 161 genes are included in Rat Genome 230 2.0 Array. This array was used to detect expression changes of genes related to ERK1/2 signalling pathway in isolated hepatocytes of rat LR, showing that 60 genes were related to hepatocytes of LR. In addition, bioinformatics and systems biology methods were used to analyse the roles of 14 above paths in regenerating hepatocytes. We found that three paths, RTK→SHC→GRB2/SOS→RAS→RAF, IntegrinΒ→FAK→RAC→PAK→RAF and GΒγ→PI3KΒγ→RAC→PAK→RAF, promoted the G1 phase progression of hepatocytes by activating ERK1/2. A further four paths, Gq→PLCΒ→PKC→SRC/PYK2→GRB2/SOS→RAS→RAF, RTK→PLCγ→PKC→SRC/PYK2→GRB2/SOS→RAS→RAF, IntegrinΒ→FAK/SRC→GRB2/SOS→RAS→RAF and IntegrinΒ→FAK→RAC→PAK→RAF, advanced the cell progression of S phase and G(2)/M checkpoint by activating ERK1/2, and so did PP1/2→Mek1/2 by decreasing the negative influence on ERK1/2. At the late phase of LR, Gαs→AC→EPAC→Rap1→Raf blocked hepatocyte proliferation by decreasing the activity of ERK1/2 and so did PP1/2→Mek1/2. In summary, 60 genes and 8 paths of ERK1/2 signalling pathway regulated hepatocyte proliferation in rat LR.

Resveratrol Inhibits Proliferation and Survival of Epstein Barr Virus–Infected Burkitt's Lymphoma Cells Depending on Viral Latency Program

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenolic natural product, shows chemopreventive properties against several cancers, heart diseases, inflammation, and viral infections. Epstein Barr virus (EBV), a γ-herpesvirus, contributes to the development of several human cancers including Burkitt's lymphoma (BL). In this study, we asked whether treatment with resveratrol would affect the viability of EBV-positive BL cells displaying different forms of latency. We report here that resveratrol, regardless of EBV status, induces caspase-dependent apoptosis by arresting cell-cycle progression in G(1) phase. However, resveratrol strongly induced apoptosis in EBV(-) and latency I EBV(+) cells, whereas latency II and latency III EBV(+) BL cells showed a survival advantage that increased with the extent of the pattern of viral gene expression. Resveratrol-induced cell-cycle arrest and apoptosis occurred in association with induction of p38 MAPK phosphorylation and suppression of ERK1/2 signaling pathway. Moreover, NF-κB DNA-binding activity was inhibited in all BL lines except EBV(+) latency III cells. LMP1 oncogene, which is expressed in latency III phenotype, is involved with the higher resistance to the antiproliferative effect of resveratrol because siRNA-mediated inhibition of LMP1 greatly increased the sensitivity of latency III BL cells as well as that of lymphoblastoid cell lines to the polyphenol. We propose that a combined resveratrol/siRNA strategy may be a novel approach for the treatment of EBV-associated B-cell malignancies in which the viral pattern of gene expression has been defined.

Phosphorylation of Cdc25B3 Ser169 regulates 14-3-3 binding to Ser151 and Cdc25B activity

Cdc25B is a major regulator of G2 phase progression into mitosis and in recovery from a G2 phase checkpoint arrest. The activity of Cdc25B is regulated by 14-3-3 dimer binding to a high-affinity phosphosite Ser323 and two lower-affinity phosphosites Ser151 and Ser230, which blocks substrate access to the catalytic site and, thereby, activity. Loss of 14-3-3 binding to any of these sites is sufficient to activate the enzyme. Thus, the mechanisms that disrupt 14-3-3 binding are critical regulators of entry into mitosis. Here, we demonstrate that the mechanism regulating the G2 phase activation of major cdc25B3 isoform is independent of the dephosphorylation of the 14-3-3 binding sites, instead utilizing phosphorylation of Ser169. Mutation of this site to the phosphomimetic Asp reduced 14-3-3 binding to the same extent as Ser151Ala mutation, increased substrate access to the catalytic site and Cdc25B activity but had no effect on Ser151 or Ser230 phosphorylation. Ser169 phosphorylation was observed in G2-phase cells. By contrast, Ser249 phosphorylation, which was associated with cyclin B/Cdk1 activation, had no effect on 14-3-3 binding or Cdc25B activity. This indicates that Ser169 phosphorylation can disrupt 14-3-3 binding to Ser151 activating Cdc25B3, providing a mechanism for regulating Cdc25B3 activation without dephosphorylation of the 14-3-3 binding sites.

GH Receptor Plays a Major Role in Liver Regeneration through the Control of EGFR and ERK1/2 Activation

GH is a pleiotropic hormone that plays a major role in proliferation, differentiation, and metabolism via its specific receptor. It has been previously suggested that GH signaling pathways are required for normal liver regeneration but the molecular mechanisms involved have yet to be determined. The aim of this study was to identify the mechanisms by which GH controls liver regeneration. We performed two thirds partial hepatectomies in GH receptor (GHR)-deficient mice and wild-type littermates and showed a blunted progression in the G(1)/S transition phase of the mutant hepatocytes. This impaired liver regeneration was not corrected by reestablishing IGF-1 expression. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between mutant and wild-type mice, cell cycle progression was significantly blunted in mutant mice. The main defect in GHR-deficient mice was the deficiency of the epidermal growth factor receptor activation during the process of liver regeneration. Finally, among the pathways activated downstream of GHR during G(1) phase progression, namely Erk1/2, Akt, and signal transducer and activator of transcription 3, we only found a reduced Erk1/2 phosphorylation in mutant mice. In conclusion, our results demonstrate that GH signaling plays a major role in liver regeneration and strongly suggest that it acts through the activation of both epidermal growth factor receptor and Erk1/2 pathways.

Human NDR Kinases Control G1/S Cell Cycle Transition by Directly Regulating p21 Stability

The G(1) phase of the cell cycle is an important integrator of internal and external cues, allowing a cell to decide whether to proliferate, differentiate, or die. Multiple protein kinases, among them the cyclin-dependent kinases (Cdks), control G(1)-phase progression and S-phase entry. With the regulation of apoptosis, centrosome duplication, and mitotic chromosome alignment downstream of the HIPPO pathway components MST1 and MST2, mammalian NDR kinases have been implicated to function in cell cycle-dependent processes. Although they are well characterized in terms of biochemical regulation and upstream signaling pathways, signaling mechanisms downstream of mammalian NDR kinases remain largely unknown. We identify here a role for human NDR in regulating the G(1)/S transition. In G(1) phase, NDR kinases are activated by a third MST kinase (MST3). Significantly, interfering with NDR and MST3 kinase expression results in G(1) arrest and subsequent proliferation defects. Furthermore, we describe the first downstream signaling mechanisms by which NDR kinases regulate cell cycle progression. Our findings suggest that NDR kinases control protein stability of the cyclin-Cdk inhibitor protein p21 by direct phosphorylation. These findings establish a novel MST3-NDR-p21 axis as an important regulator of G(1)/S progression of mammalian cells.

Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: The critical role of hEag1 channels in G1 phase progression

Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K+) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K+ channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K+ channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21WAF1/Cip1 expression with a kinetic similar to that of cyclin D1, however p27Kip1 expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6–8h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21WAF1/Cip1 and p27Kip1 expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K+ channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E.

G 1 /S Transcription Factor Orthologues Swi4p and Swi6p Are Important but Not Essential for Cell Proliferation and Influence Hyphal Development in the Fungal Pathogen Candida albicans

The G(1)/S transition is a critical control point for cell proliferation and involves essential transcription complexes termed SBF and MBF in Saccharomyces cerevisiae or MBF in Schizosaccharomyces pombe. In the fungal pathogen Candida albicans, G(1)/S regulation is not clear. To gain more insight into the G(1)/S circuitry, we characterized Swi6p, Swi4p and Mbp1p, the closest orthologues of SBF (Swi6p and Swi4p) and MBF (Swi6p and Mbp1p) components in S. cerevisiae. The mbp1Δ/Δ cells showed minor growth defects, whereas swi4Δ/Δ and swi6Δ/Δ yeast cells dramatically increased in size, suggesting a G(1) phase delay. Gene set enrichment analysis (GSEA) of transcription profiles revealed that genes associated with G(1)/S phase were significantly enriched in cells lacking Swi4p and Swi6p. These expression patterns suggested that Swi4p and Swi6p have repressing as well as activating activity. Intriguingly, swi4Δ/Δ swi6Δ/Δ and swi4Δ/Δ mbp1Δ/Δ strains were viable, in contrast to the situation in S. cerevisiae, and showed pleiotropic phenotypes that included multibudded yeast, pseudohyphae, and intriguingly, true hyphae. Consistently, GSEA identified strong enrichment of genes that are normally modulated during C. albicans-host cell interactions. Since Swi4p and Swi6p influence G(1) phase progression and SBF binding sites are lacking in the C. albicans genome, these factors may contribute to MBF activity. Overall, the data suggest that the putative G(1)/S regulatory machinery of C. albicans contains novel features and underscore the existence of a relationship between G(1) phase and morphogenetic switching, including hyphal development, in the pathogen.

Dilong: Role in Peripheral Nerve Regeneration

Dilong, also known as earthworm, has been widely used in traditional Chinese medicine (TCM) for thousands of years. Schwann cell migration and proliferation are critical for the regeneration of injured nerves and Schwann cells provide an essentially supportive role for neuron regeneration. However, the molecular mechanisms of migration and proliferation induced by dilongs in Schwann cells remain unclear. Here, we discuss the molecular mechanisms that includes (i) migration signaling, MAPKs (mitogen-activated protein kinases), mediated PAs and MMP2/9 pathway; (ii) survival and proliferative signaling, IGF-I (insulin-like growth factor-I)-mediated PI3K/Akt pathways and (iii) cell cycle regulation. Dilong stimulate RSC96 cell proliferation and migration. It can induce phosphorylation of ERK1/2 and p38, but not JNK, and activate the downstream signaling expression of PAs (plasminogen activators) and MMPs (matrix metalloproteinases) in a time-dependent manner. In addition, Dilong stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with chemical inhibitors (U0126 and SB203580), and small interfering ERK1/2 and p38 RNA, resulting in migration and uPA-related signal pathway inhibition. Dilong also induces the phosphorylation of IGF-I-mediated PI3K/Akt pathway, activates protein expression of PCNA (proliferating cell nuclear antigen) and cell cycle regulatory proteins (cyclin D1, cyclin E and cyclin A) in a time-dependent manner. In addition, it accelerates G1-phase progression with earlier S-phase entry and significant numbers of cells entered the S-phase. The siRNA-mediated knockdown of PI3K that significantly reduces PI3K protein expression levels, resulting in Bcl2 survival factor reduction, revealing a marked blockage of G1 to S transition in proliferating cells. These results reveal the unknown RSC96 cell migration and proliferation mechanism induced by dilong, which find use as a new medicine for nerve regeneration.

The Forkhead factor FoxQ1 influences epithelial differentiation

The Forkhead family of transcription factors comprises numerous members and is implicated in various cellular functions, including cell growth, apoptosis, migration, and differentiation. In this study, we identified the Forkhead factor FoxQ1 as increased in expression during TGF-β1 induced changes in epithelial differentiation, suggesting functional roles of FoxQ1 for epithelial plasticity. The repression of FoxQ1 in mammary epithelial cells led to a change in cell morphology characterized by an increase in cell size, pronounced cell-cell contacts, and an increased expression of several junction proteins (e.g., E-cadherin). In addition, FoxQ1 knock-down cells revealed rearrangements in the actin-cytoskeleton and slowed down cell cycle G1-phase progression. Furthermore, repression of FoxQ1 enhanced the migratory capacity of coherent mammary epithelial cells. Gene expression profiling of NM18 cells indicated that FoxQ1 is a relevant downstream mediator of TGF-β1-induced gene expression changes. This included the differential expression of transcription factors involved in epithelial plasticity, for example, Ets-1, Zeb1, and Zeb2. In summary, this study has elucidated the functional impact of FoxQ1 on epithelial differentiation.

Modulation of Multiple Myeloma Apoptosis and Adhesion by Seliciclib, A Selective Inhibitor of Cyclin Dependent Kinase (CDK)

AbstractAbstract 3971Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cells that eventually develop resistance to chemotherapy. Novel agents such as Thalidomid, Bortezomib and Lenalidomide improve response rate and prolong progression free and overall survival. Drug resistance, differentiation block and increased survival of the MM tumor cells result from genomic alterations including high levels of cyclin D. In addition, MM displays a high genomic instability. The most common translocations in MM lead to dysregulation of cyclin D (CCND), a regulatory protein that governs the activation of a key cell cycle regulator – cyclin dependent kinase (CDK). Cell-proliferation is tightly regulated by activity of CDKs at checkpoints located at the boundaries of cell cycle stages. The association of CDK4/6 with CCND is critical for G1 phase progression, whereas CDK2-cycllin E is necessary for S phase initiation. Deviation from the orderly control may lead to malignant transformation. Furthermore, genomic instability was reported to be affected by over expression of cyclin E. Cyclin E deregulation induces genomic instability as an early event in tumorgenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore, sought to investigate the role of cyclin E in MM. We first examined the expression profile of CCNE1 in various MM cell lines (hMMCLs) including ArP-1, ARH77, CAG, NCIH929, RPMI8226 and U266 by immunoblotting utilizing CCNE1 Abs and by qRT-PCR. CCNE1 expression was found to be heterogeneous being high in U266 and NCIH292, intermediate in ARH77, CAG and PRMI8226 and low in ArP-1 MM cell lines, respectively. A direct correlation between the level of protein and the transcript was observed. We then evaluated the effect of Seliciclib (Roscovitine, CYC202), a selective inhibitor of CDK's on hMMCLs viability, using the MTT assay as hMMCLs have been reported to be sensitive to CDK inhibition. The various hMMCLs were incubated with increasing concentration of Seliciclib (0-100 μM) for 3 days. Seliciclib inhibition of CDK resulted in dose-dependent cytotoxicity with IC50 of 20–80 μM with PRMI8226 being the most sensitive, while ARH77 hMMCL the most resistant. Cell cycle study demonstrated increased apoptosis (based on sub G1 and annexin V analysis) in the sensitive hMMCLs, with no effect on the resistant cell lines. Annexin V binding was observed within 5h and reached maximal level 12h flowing Seliciclib (50 μM) exposure. We next assessed the effect Seliciclib on MCL1, an anti-apoptotic BCL2 family member that is consistitutively expressed in MM cells. Seliciclib significantly down-regulated MCL1 expression in a time and dose dependant manner. Assessing the effect of Seliciclib on cell cycle regulators including CCND, CCNE and P27kip1 indicated a very significant reduction in CCND2 and P27 expression levels in most of the hMMCLs in a dose and time dependant manner, while CCNE1 expression levels increased. Transfection of the low expressing hMMCL RPMI8226 with CCNE1, inducing over expression of cyclin E, resulted in reduced sensitivity of the MM tumor cells to the Seliciclib induced cell death in comparison to the paternal cell line. Lastly, we investigated the effect of Seliciclib on MM cell adhesion to fibronectin (FN), as cell-adhesion-mediated-drug-resistance (CAM-DR) is a major importance protecting MM tumor cells from cytotoxic drugs induced apoptosis. hMMCLs were pre-incubated with increasing concentration of Seliciclib for 1h, plated onto 40 μg/ml FN coated plates for 1h, washed and stained by crystal violet. Seliciclib resulted in a direct dose dependant inhibition of adhesion. The adhesive protective property was evalued by CAM-DR assay, whereas FN-adhesion protected the MM tumor cells against doxorubicin mediated cytotoxicity. There was no difference between the cytotoxic effects of Seliciclib on FN-MM adherent vs. suspended tumor cells. In summary incubation of MM tumor cells with Seliciclib, a selective inhibitor of CDK's, results in apoptosis which is mediated by down regulation of MCL1 and is accompanied by reduced adhesion to FN. Its ability to prevent adhesion – mediated – drug resistance of MM tumor cells may turn Seliciclib as essential component of modern anti MM drug combination therapy. Further experiments should test its synergism with anti MM novel agents. Disclosures:No relevant conflicts of interest to declare.