NORMAL GENOMIC

• Capasso S et al.
• Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.
• Cell Cycle. 2014; 13: 482-90
• Display abstract

• Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation. Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets. Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis. Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.

• Liu Q et al.
• Amphiregulin stimulates liver regeneration after small-for-size mouse liver transplantation.
• Am J Transplant. 2012; 12: 2052-61
• Display abstract

• This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small-for-size liver transplantation. Livers of male C57BL/6 mice were reduced to ~50% and ~30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5-Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 mug/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small-for-size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.

• Machado CF et al.
• Disease-associated mutations in the prion protein impair laminin-induced process outgrowth and survival.
• J Biol Chem. 2012; 287: 43777-88
• Display abstract

• Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrP(C)) to propagate disease. PrP(C) is converted into an abnormal insoluble form, PrP(Sc), that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrP(Sc) deposits, but the loss of normal PrP(C) function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrP(C) represent one of the best models to understand the impact of PrP(C) loss-of-function. PrP(C) associates with various molecules and, in particular, the interaction of PrP(C) with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrP(C) mutations, wild-type and mutated PrP(C) proteins were expressed in a neural cell line derived from a PrP(C)-null mouse. Treatment with the laminin gamma1 chain peptide (Ln gamma1), which mimics the Ln binding site for PrP(C), increased intracellular calcium in cells expressing wild-type PrP(C), whereas a significantly lower response was observed in cells expressing mutated PrP(C) molecules. The Ln gamma1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrP(C) molecules in contrast to cells expressing wild-type PrP(C). The co-expression of wild-type PrP(C) with mutated PrP(C) molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrP(C) mutated molecules. These results indicate that PrP(C) mutations impair process outgrowth and survival mediated by Ln gamma1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases.

• Zhou QM, Wang XF, Liu XJ, Zhang H, Lu YY, Su SB
• Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo.
• Acta Pharmacol Sin. 2011; 32: 1402-10
• Display abstract

• AIM: To investigate the efficacy of mitomycin C (MMC) in combination with curcumin in suppressing human breast cancer in vitro and in vivo. METHODS: Human breast cancer MCF-7 cells were used. Cell viability was measured using MTT assay. The cell cycle phase was detected with flow cytometric analysis. Cell cycle-associated proteins were examined using Western blot analysis. MCF-7 breast cancer xenografts were established to monitor tumor growth and cell cycle-associated protein expression. RESULTS: Curcumin inhibited MCF-7 breast cancer cell viability in a concentration-dependent manner (IC(50) value=40 mumol/L). Similarly, MMC inhibited the cell viability with an IC(50) value of 5 mumol/L. Combined treatment of MMC and curcumin showed a synergistic antiproliferative effect. In the presence of curcumin (40 mumol/L), the IC(50) value of MMC was reduced to 5 mumol/L. In MCF-7 xenografts, combined administration of curcumin (100 mg/kg) and MMC (1-2 mg/kg) for 4 weeks produced significantly greater inhibition on tumor growth than either treatment alone. The combined treatment resulted in significantly greater G(1) arrest than MMC or curcumin alone. Moreover, the cell cycle arrest was associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2) and CDK4, along with the induction of the cell cycle inhibitor p21 and p27 both in MCF-7 cells and in MCF-7 xenografts. These proteins were regulated through p38 MAPK pathway. CONCLUSION: The results suggest that the combination of MMC and curcumin inhibits MCF-7 cell proliferation and cell cycle progression in vitro and in vivo via the p38 MAPK pathway.

• Zang Y, Yu LF, Nan FJ, Feng LY, Li J
• AMP-activated protein kinase is involved in neural stem cell growth suppression and cell cycle arrest by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and glucose deprivation by down-regulating phospho-retinoblastoma protein and cyclin D.
• J Biol Chem. 2009; 284: 6175-84
• Display abstract

• The fate of neural stem cells (NSCs), including their proliferation, differentiation, survival, and death, is regulated by multiple intrinsic signals and the extrinsic environment. We had previously reported that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) directly induces astroglial differentiation of NSCs by activation of the Janus kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway independently of AMP-activated protein kinase (AMPK). Here, we reported the observation that AICAR inhibited NSC proliferation and its underlying mechanism. Analysis of caspase activity and cell cycle showed that AICAR induced G1/G0 cell cycle arrest in NSCs, associated with decreased levels of poly(ADP-ribose) polymerase, phospho-retinoblastoma protein (Rb), and cyclin D but did not cause apoptosis. Iodotubericidin and Compound C, inhibitors of adenosine kinase and AMPK, respectively, or overexpression of a dominant-negative mutant of AMPK, but not JAK inhibitor, were able to reverse the anti-proliferative effect of AICAR. Glucose deprivation also activated the AMPK pathway, induced G0/G1 arrest, and suppressed the proliferation of NSCs, an effect associated with decreased levels of phospho-Rb and cyclin D protein. Furthermore, Compound C and overexpression of dominant-negative AMPK in C17.2 NSCs could block the glucose deprivation-mediated down-regulation of cyclin D and partially reverse the suppression of proliferation. These results suggest that AICAR and glucose deprivation might induce G1/G0 cell cycle arrest and suppress proliferation of NSCs via phospho-Rb and cyclin D down-regulation. AMPK, but not JAK/STAT3, activation is key for this inhibitory effect and may play an important role in the responses of NSCs to metabolic stresses such as glucose deprivation.

• Stoica BA, Byrnes KR, Faden AI
• Cell cycle activation and CNS injury.
• Neurotox Res. 2009; 16: 221-37
• Display abstract

• Although traditionally it was believed that adult neurons have entered a permanent post-mitotic phase, the latest experimental data indicate that cell cycle constituents critically affect normal functions of the adult central nervous system (CNS), as well as contribute to the pathophysiology of both acute and chronic neurodegenerative disorders. Recent study has also suggested that cell cycle pathways are involved in mediating not only neuronal cell death but also glial changes which may play key roles in the pathophysiological mechanisms underlying acute and chronic neurodegenerative disorders. Therefore, therapies that inhibit cell cycle may have robust neuroprotective profiles, particularly after acute insults, by targeting multiple pathogenic mechanisms and could be useful clinically if used with a delivery focused on specific areas and of a limited duration.

• Schmetsdorf S, Arnold E, Holzer M, Arendt T, Gartner U
• A putative role for cell cycle-related proteins in microtubule-based neuroplasticity.
• Eur J Neurosci. 2009; 29: 1096-107
• Display abstract

• Cyclins and cyclin-dependent kinases (Cdks) are the main components that control the orderly progression through cell cycle. In the mature nervous system, terminally differentiated neurons are permanently withdrawn from cell cycle, as mitotic quiescence is essential for the functional stability of the complexly wired neuronal system. Recently, we characterized the expression and colocalization of cyclins and Cdks in terminally differentiated pyramidal neurons. The functional impact of the expression of cell cycle-related proteins in differentiated neurons, however, has not been elucidated yet. In the present study, we show by immunoelectron microscopy and immunobiochemical methods an association of cyclins and Cdks with the microtubule network. Cyclins D, E, A and B as well as Cdks 1, 2 and 4 were also found to be associated with the microtubule-associated protein tau. Cyclin/Cdk complexes, in addition, exhibit kinase activity towards tau. In vitro, downregulation of cyclins and Cdks by a siRNA approach and by pharmacological inhibition promotes neurite extension. Taken together, these results indicate that the expression of cell cycle-related proteins in terminal differentiated neurons is associated with physiological functions beyond cell cycle control that might be involved in microtubule-based mechanisms of neuroplasticity.

• Georgopoulou N, Hurel C, Politis PK, Gaitanou M, Matsas R, Thomaidou D
• BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation.
• J Biol Chem. 2006; 281: 33606-20
• Display abstract

• Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G(1) phase, most probably corresponding to cell cycle exit at the G(0) restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.

• Andrieu D, Meziane H, Marly F, Angelats C, Fernandez PA, Muscatelli F
• Sensory defects in Necdin deficient mice result from a loss of sensory neurons correlated within an increase of developmental programmed cell death.
• BMC Dev Biol. 2006; 6: 56-56
• Display abstract

• BACKGROUND: The human NECDIN gene is involved in a neurodevelopmental disorder, Prader-Willi syndrome (PWS). Previously we reported a mouse Necdin knock-out model with similar defects to PWS patients. Despite the putative roles attributed to Necdin, mainly from in vitro studies, its in vivo function remains unclear. In this study, we investigate sensory-motor behaviour in Necdin deficient mice. We reveal cellular defects and analyse their cause. RESULTS: We report sensory differences in Necdin deficient mice compared to wild type animals. These differences led us to investigate sensory neuron development in Necdin deficient mouse embryos. First, we describe the expression pattern of Necdin in developing DRGs and report a reduction of one-third in specified sensory neurons in dorsal roots ganglia and show that this neuronal loss is achieved by E13.5, when DRGs sensory neurons are specified. In parallel, we observed an increase of 41% in neuronal apoptosis during the wave of naturally occurring cell death at E12.5. Since it is assumed that Necdin is a P75NTR interactor, we looked at the P75NTR-expressing cell population in Necdin knock-out embryos. Unexpectedly, Necdin loss of function has no effect on p75NTR expressing neurons suggesting no direct genetic interaction between Necdin and P75NTR in this context. Although we exclude a role of Necdin in axonal outgrowth from spinal sensory neurons in early developmental stages; such a role could occur later in neuronal differentiation. Finally we also exclude an anti-proliferative role of Necdin in developing sensory neurons. CONCLUSION: Overall, our data show clearly that, in early development of the nervous system, Necdin is an anti-apoptotic or survival factor.

• Mawson A, Lai A, Carroll JS, Sergio CM, Mitchell CJ, Sarcevic B
• Estrogen and insulin/IGF-1 cooperatively stimulate cell cycle progression in MCF-7 breast cancer cells through differential regulation of c-Myc and cyclin D1.
• Mol Cell Endocrinol. 2005; 229: 161-73
• Display abstract

• Estrogen and insulin/insulin-like growth factor-I (IGF-I) are major mitogens for breast epithelial cells and when co-administered, synergistically induce G(1)-S phase cell cycle progression. We investigated this cooperativity by evaluating if the key cell cycle regulators, c-Myc and cyclin D1, represent points of convergence in the action of these mitogens in MCF-7 breast cancer cells. These studies demonstrated that estrogen significantly increased both c-Myc and cyclin D1 protein, while insulin predominantly increased cyclin D1 levels. This cumulative increase in c-Myc and cyclin D1 contributes to the cooperativity of these mitogens, since ectopic expression of c-Myc or cyclin D1 cooperates with either the estrogen or insulin signaling pathways to increase cell cycle progression. Inhibition of the MAPK or PI3-kinase pathways significantly reduced c-Myc and cyclin D1 protein levels and cell cycle progression. Ectopic expression of cyclin D1 partially overcame this inhibition, while ectopic expression of c-Myc partially overcame MAPK but not PI3-kinase inhibition. Therefore, estrogen and insulin/IGF-1 differentially regulate c-Myc and cyclin D1 to cooperatively stimulate breast cancer cell proliferation.