• Genes
• Genetic bases
• Grooming
• In silico modeling
• Molecular network
• Self-grooming

• Mice
• Animals
• Grooming/physiology
• Neurons

• CSB
• Cockayne syndrome
• Necdin
• Neurodegeneration
• Transcription regulation

• schizophrenia
• clinical genetics

Necdin was originally found in 1991 as a hypothetical protein encoded by a neural differentiation‐specific gene transcript in murine embryonal carcinoma cells. Virtually all postmitotic neurons and their precursor cells express the necdin gene (Ndn) during neuronal development. Necdin mRNA is expressed only from the paternal allele through genomic imprinting, a placental mammal‐specific epigenetic mechanism. Necdin and its homologous MAGE (melanoma antigen) family, which have evolved presumedly from a subcomplex component of the SMC5/6 complex, are expressed exclusively in placental mammals. Paternal Ndn‐mutated mice totally lack necdin expression and exhibit various types of neuronal abnormalities throughout the nervous system. Ndn‐null neurons are vulnerable to detrimental stresses such as DNA damage. Necdin also suppresses both proliferation and apoptosis of neural stem/progenitor cells. Functional analyses using Ndn‐manipulated cells reveal that necdin consistently exerts antimitotic, anti‐apoptotic and prosurvival effects. Necdin interacts directly with a number of regulatory proteins including E2F1, p53, neurotrophin receptors, Sirt1 and PGC‐1α, which serve as major hubs of protein–protein interaction networks for mitosis, apoptosis, differentiation, neuroprotection and energy homeostasis. This review focuses on necdin as a pleiotropic protein that integrates molecular interaction networks to promote neuronal vitality in modern placental mammals.

• DNA damage response
• MAGE family
• Prader-Willi syndrome
• genomic imprinting
• mammalian brain
• necdin
• neuronal development
• neuronal vitality
• neuroprotection
• protein-protein interaction network

• AMP-activated kinase (AMPK)
• DNA damage response
• E3 ligase
• E3 ubiquitin ligase
• Fe-S cluster
• MAGE
• Prader-Willi syndrome
• Schaaf-Yang syndrome
• alternative polyadenylation
• apoptosis
• cancer
• cancer-testis antigen
• cell metabolism
• melanoma antigen
• metabolism
• p53
• spermatogenesis
• stress adaptation
• stress granule
• stress response
• ubiquitin
• ubiquitination

• Animals
• Antigens, Neoplasm/genetics
• Antigens, Neoplasm/metabolism
• Gene Expression Regulation, Neoplastic
• Humans
• Neoplasm Proteins/genetics
• Neoplasm Proteins/metabolism
• Neoplasms/genetics
• Neoplasms/metabolism
• Stress, Physiological
• Ubiquitin-Protein Ligases/genetics
• Ubiquitin-Protein Ligases/metabolism

• R01 GM111332 NIGMS NIH HHS

• ARNTL Transcription Factors/metabolism
• Adaptor Proteins, Signal Transducing/metabolism
• Animals
• Cell Cycle Proteins/metabolism
• Circadian Clocks/genetics
• Gene Expression
• HSP90 Heat-Shock Proteins/metabolism
• Mice
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/metabolism
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Proteasome Endopeptidase Complex/metabolism
• Protein Stability
• Ubiquitin/metabolism

• National Natural Science Foundation of China
• Hunan Province
• Guangdong Key Project
• Central South University
• Central Universities of Central South University

• E2f
• Ect2
• Rb
• binucleated
• cardiac regeneration
• cardiomyocyte
• development
• heart
• mononucleated
• proliferation

• Animals
• Animals, Newborn
• Base Sequence
• Cell Nucleus/metabolism
• Cell Nucleus/ultrastructure
• Cell Proliferation
• Cell Separation
• Down-Regulation/genetics
• E2F Transcription Factors/metabolism
• Flow Cytometry
• G1 Phase
• Mice, Knockout
• Myocytes, Cardiac/cytology
• Myocytes, Cardiac/metabolism
• Myocytes, Cardiac/ultrastructure
• Proto-Oncogene Proteins/metabolism
• Regeneration
• Retinoblastoma Protein/metabolism
• S Phase

• T32 DK007780 NIDDK NIH HHS
• U01 HL100405 NHLBI NIH HHS
• T32 HL007954 NHLBI NIH HHS
• T32 GM007229 NIGMS NIH HHS
• U01 HL134745 NHLBI NIH HHS

• Neural stem cells
• Neuronal differentiation
• Prader-Willi syndrome
• iPS cells

Exosomes have been regarded as emerging tools for cancer diagnosis. Tumor-derived exosomes contain molecules that enhance cancer progression and affect immune responses.

In the present study, we evaluated expression of seven cancer-testis antigens (CTAs) that are regarded as putative biomarkers and immunotherapeutic targets along with NMP22 in urinary exosomes of bladder cancer patients, healthy subjects and patients affected with nonmalignant urinary disorders.

Exosomal expression of MAGE-B4 was significantly higher in bladder cancer patients compared with normal samples (expression ratio=2.68, P=0.01). However, its expression was lower in bladder cancer patients compared with benign prostate hyperplasia (BPH) patients (expression ratio=0.17, P=0.01). Exosomal expression of NMP22 was significantly higher in bladder cancer patients compared with BPH patients (expression ratio=9.22, P=0.02). Expressions of other genes were not significantly different between bladder cancer patients and normal/nonmalignant samples. We found significant correlation between MAGE-A3 and MAGE-B4 expressions in exosomes obtained from controls. In addition, TSGA10 expression was correlated with expression of NMP22 in both cancer patients and controls.

The present study provides evidences for differential expression of CTAs in urinary exosomes of bladder cancer patients and urogenital disorders and warrants further studies for assessment of their significance in cancer diagnosis and immunotherapeutic approaches.

• bladder cancer
• cancer-testis antigen
• exosome

The progression of CRC is a multistep process involving several genetic changes or epigenetic modifications. NDN is a member of the MAGE family, encoding a protein that generally suppresses cell proliferation and acting as a transcriptional repressor. Immunohistochemical staining revealed that the expression of NDN was significantly down-regulated in CRC tissues compared with normal tissues and the down-regulation of NDN in CRC could reflect the hypermethylation of the NDN promoter. Treatment of the CRC cell line SW480 with the demethylating agent 5-Aza-CdR restored the NDN expression level. The down-regulation of NDN was closely related to poor differentiation, advanced TNM stage and poor prognosis of CRC. The inhibition of NDN promoted CRC cell proliferation by enriching cells in the S phase. Furthermore, we observed that NDN binds to the GN box in the promoter of LRP6 to attenuate LRP6 transcription and inhibit the Wnt signaling pathway in CRC. In conclusion, our study revealed that the hypermethylation of NDN promotes cell proliferation by activating the Wnt signaling pathway through directly increasing the transcription of LRP6 in CRC. These findings might provide a new theoretical basis for the pathogenesis of CRC and facilitate the development of new therapeutic strategies against CRC.

• LRP6
• NDN
• Wnt signaling pathway
• colorectal cancer
• proliferation

Acute myeloid leukemia (AML) is a devastating illness which carries a very poor prognosis, with most patients living less than 18 months. Leukemia relapse may occur because current therapies eliminate proliferating leukemia cells but fail to eradicate quiescent leukemia-initiating cells (LICs) that can reinitiate the disease after a period of latency. While we demonstrated that p53 target gene Necdin maintains hematopoietic stem cell (HSC) quiescence, its roles in LIC quiescence and response to chemotherapy are unclear. In this study, we utilized two well-established murine models of human AML induced by MLL-AF9 or AML1-ETO9a to determine the role of Necdin in leukemogenesis. We found that loss of Necdin decreased the number of functional LICs and enhanced myeloid differentiation in vivo, leading to delayed development of leukemia induced by MLL-AF9. Importantly, Necdin null LICs expressing MLL-AF9 were less quiescent than wild-type LICs. Further, loss of Necdin enhanced the response of MLL-AF9⁺ leukemia cells to chemotherapy treatment, manifested by decreased viability and enhanced apoptosis. We observed decreased expression of Bcl2 and increased expression of p53 and its target gene Bax in Necdin null leukemia cells following chemotherapy treatment, indicating that p53-dependent apoptotic pathways may be activated in the absence of Necdin. In addition, we found that loss of Necdin decreased the engraftment of AML1-ETO9a⁺ hematopoietic stem and progenitor cells in transplantation assays. However, Necdin-deficiency did not affect the response of AML1-ETO9a⁺ hematopoietic cells to chemotherapy treatment. Thus, Necdin regulates leukemia-initiating cell quiescence and chemotherapy response in a context-dependent manner. Our findings suggest that pharmacological inhibition of Necdin may hold potential as a novel therapy for leukemia patients with MLL translocations.

• MLL-AF9
• Necdin
• chemotherapy
• leukemia-initiating cells
• quiescence

NDN is a maternally imprinted gene consistently expressed in normal ovarian epithelium, is dramatically downregulated in the majority of ovarian cancers. Little or no NDN expression could be detected in 73% of 351 epithelial ovarian cancers. NDN was also downregulated in 10 ovarian cancer cell lines with total loss in 6 of 10. Re-expression of NDN decreased Bcl-2 levels and induced apoptosis, which significantly inhibited ovarian cancer cell growth in cell culture and in xenografts. In addition, re-expression of NDN inhibited cell migration by decreasing actin stress fiber and focal adhesion complex formation through deactivation of Src, FAK and RhoA. Loss of NDN expression in ovarian cancers could be attributed to LOH in 28% of 18 informative cases and to hypermethylation of CpG sites 1 and 2 of NDN promoter in 23% and 30% of 43 ovarian cancers, respectively. Promoter hypermethylation was also found in 5 of 10 ovarian cancer cell lines. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored NDN expression in 4 of 7 cell lines with enhanced promoter methylation levels. These observations support the conclusion that NDN is an imprinted tumor suppressor gene which affects cancer cell motility, invasion and growth and that its loss of function in ovarian cancer can be caused by both genetic and epigenetic mechanisms.

• FAK
• NDN
• cell motility
• growth inhibition
• imprinting
• tumor suppressor gene

Metastasis is the primary cause of death in breast cancer. Earlier studies using a mammary tumorigenesis mouse model identified Necdin (Ndn) as a germline modifier of metastasis. Differential expression of Ndn induces a gene-expression signature that predicts prognosis in human breast cancer. Additionally, a non-synonymous germline single nucleotide polymorphism (T50C; V17A) in Ndn distinguishes mouse strains with differing metastatic capacities. To better understand how hereditary factors influence metastasis in breast cancer, we characterized NDN-mediated transcription. Haplotype analysis in a well-characterized breast cancer cohort revealed that NDN germline variation is associated with both NDN expression levels and patient outcome. To examine the role of NDN in mammary tumor metastasis and transcriptional regulation, mouse mammary tumor cell lines stably over-expressing either the wildtype 50T or variant 50C Ndn allele were generated. Cells over-expressing Ndn 50T, but not Ndn 50C, exhibited significant decrease in cell invasiveness and pulmonary metastases compared to control cells. Transcriptome analyses identified a 71-gene expression signature that distinguishes cells over-expressing the two Ndn allelic variants. Furthermore, ChIP assays revealed c-Myc, a target gene of NDN, to be differentially regulated by the allelic variants. These data demonstrate that NDN and the T50C allele regulate gene expression and metastasis efficiency.

• breast cancer
• c-Myc
• germline polymorphisms
• metastasis suppressors
• necdin

• NDN gene
• Necdin
• Prognosis.
• Urothelial carcinoma

• EGFR
• ERK
• Gliogenesis
• MAGE family
• Necdin

• E2F transcription factor
• cancer biology
• cell proliferation
• histone deacetylase 1 (HDAC1)
• molecular cell biology

Neural stem cells (NSCs) and imprinted genes play an important role in brain development. On historical grounds, these two determinants have been largely studied independently of each other. Recent evidence suggests, however, that NSCs can reset select genomic imprints to prevent precocious depletion of the stem cell reservoir. Moreover, imprinted genes like the transcriptional regulator Zac1 can fine tune neuronal vs astroglial differentiation of NSCs. Zac1 binds in a sequence-specific manner to pro-neuronal and imprinted genes to confer transcriptional regulation and furthermore coregulates members of the p53-family in NSCs. At the genome scale, Zac1 is a central hub of an imprinted gene network comprising genes with an important role for NSC quiescence, proliferation and differentiation. Overall, transcriptional, epigenomic, and genomic mechanisms seem to coordinate the functional relationships of NSCs and imprinted genes from development to maturation, and possibly aging.

• Zac1
• Cell fate decisions
• Neural stem cells
• Genomic imprinting
• Igf2-H19
• Dlk1
• p57^Kip2
• Necdin
• Differentiation
• Imprinted gene networks

Heterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous.

A neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components.

These results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor.

Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation.

• Animals
• Cell Line
• Humans
• Melanoma-Specific Antigens/metabolism
• Mice
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/metabolism
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Proteasome Endopeptidase Complex/metabolism
• Protein Binding
• Protein Inhibitors of Activated STAT/chemistry
• Protein Inhibitors of Activated STAT/metabolism
• Protein Interaction Domains and Motifs
• Proteolysis
• Substrate Specificity
• Sumoylation
• Ubiquitin/metabolism
• Ubiquitin-Protein Ligases/metabolism
• Ubiquitination

Neural precursor cells (NPCs) in the neocortex exhibit a high proliferation capacity during early embryonic development and give rise to cortical projection neurons after maturation. Necdin, a mammal-specific MAGE (melanoma antigen) family protein that possesses anti-mitotic and pro-survival activities, is expressed abundantly in postmitotic neurons and moderately in tissue-specific stem cells or progenitors. Necdin interacts with E2F transcription factors and suppresses E2F1-dependent transcriptional activation of the cyclin-dependent kinase Cdk1 gene. Here we show that necdin serves as a suppressor of NPC proliferation in the embryonic neocortex. Necdin is moderately expressed in the ventricular zone of mouse embryonic neocortex, in which proliferative cell populations are significantly increased in necdin-null mice. In the neocortex of necdin-null embryos, expression of Cdk1 and Sox2, a stem cell marker, is significantly increased, whereas expression of p16, a cyclin-dependent kinase inhibitor, is markedly diminished. Cdk1 and p16 expression levels are also significantly increased and decreased, respectively, in primary NPCs prepared from necdin-null embryos. Intriguingly, necdin interacts directly with Bmi1, a Polycomb group protein that suppresses p16 expression and promotes NPC proliferation. In HEK293A cells transfected with luciferase reporter constructs, necdin relieves Bmi1-dependent repression of p16 promoter activity, whereas Bmi1 counteracts necdin-mediated repression of E2F1-dependent Cdk1 promoter activity. In lentivirus-infected primary NPCs, necdin overexpression increases p16 expression, suppresses Cdk1 expression, and inhibits NPC proliferation, whereas Bmi1 overexpression suppresses p16 expression, increases Cdk1 expression, and promotes NPC proliferation. Our data suggest that embryonic NPC proliferation in the neocortex is regulated by the antagonistic interplay between necdin and Bmi1.

• Animals
• Cell Cycle Proteins/genetics
• Cell Cycle Proteins/metabolism
• Cell Proliferation
• Cyclin-Dependent Kinase Inhibitor p16/genetics
• Cyclin-Dependent Kinase Inhibitor p21/genetics
• Embryo, Mammalian/metabolism
• Female
• Gene Expression
• Gene Expression Regulation, Developmental
• Humans
• Male
• Mice
• Mice, Knockout
• Neocortex/cytology
• Neocortex/embryology
• Neocortex/metabolism
• Nerve Tissue Proteins/deficiency
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/metabolism
• Neural Stem Cells/metabolism
• Nuclear Proteins/deficiency
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Polycomb Repressive Complex 1/metabolism
• Promoter Regions, Genetic
• Protein Binding
• Proto-Oncogene Proteins/metabolism

Cystin is a novel cilia-associated protein that is disrupted in the cpk mouse, a well-characterized mouse model of autosomal recessive polycystic kidney disease (ARPKD). Interestingly, overexpression of the Myc gene is evident in animal models of ARPKD and is thought to contribute to the renal cystic phenotype. Using a yeast two-hybrid approach, the growth suppressor protein necdin, known to modulate Myc expression, was found as an interacting partner of cystin. Deletion mapping demonstrated that the C-terminus of cystin and both termini of necdin are required for their mutual interaction. Speculating that these two proteins may function to regulate gene expression, we developed a luciferase reporter assay and observed that necdin strongly activated the Myc P1 promoter, and cystin did so more modestly. Interestingly, the necdin effect was significantly abrogated when cystin was co-transfected. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed a physical interaction with both necdin and cystin and the Myc P1 promoter, as well as between these proteins. The data suggest that these proteins likely function in a regulatory complex. Thus, we speculate that Myc overexpression in the cpk kidney results from the dysregulation of the cystin-necdin regulatory complex and c-Myc, in turn, contributes to cystogenesis in the cpk mouse.

• Androgen Receptor
• Cell Cycle Regulation
• E2F Transcription Factor
• Gene Transcription
• MAGE-A11
• Melanoma Antigen-A11
• Prostate Cancer
• Retinoblastoma (Rb)
• Steroid Receptor Coregulator
• p107

Necdin (NDN) expression is downregulated in telomerase-immortalised normal human urothelial cells. Telomerase-immortalised normal human urothelial cells have no detected genetic alterations. Accordingly, many of the genes whose expression is altered following immortalisation are those for which epigenetic silencing is reported.

NDN expression was examined in normal tissues and tumour cell lines by quantitative real-time PCR and immunoblotting. Immunohistochemistry was performed on urothelial carcinoma (UC). Urothelial carcinoma and UC cell lines were subject to HumanMethylation27 BeadChip Array-based methylation analyses. Mutation screening was performed. The functional significance of NDN expression was investigated using retroviral-mediated downregulation or overexpression.

NDN protein was widely expressed in normal tissues. Loss of expression was observed in 38 out of 44 (86%) of UC cell lines and 19 out of 25 (76%) of non-UC cell lines. Loss of NDN protein was found in the majority of primary UC. Oncomine analysis demonstrated downregulation of expression in multiple tumour types. In UC, tumour-specific hypermethylation of NDN and key CpG sites where hypermethylation correlated with reduced expression were identified. Six novel mutations, including some of predicted functional significance, were identified in colorectal and ovarian cancer cell lines. Functional studies showed that NDN could suppress colony formation at low cell density and affect anchorage-independent growth and anoikis in vitro.

NDN is a novel tumour suppressor candidate that is downregulated and hypermethylated or mutated in cancer.

• Chromosomes, Human, Pair 15/genetics
• Chromosomes, Human, Pair 15/metabolism
• Genomic Imprinting
• Humans
• RNA/genetics
• RNA/metabolism

• R01 HD068730 NICHD NIH HHS
• R01HD068730 NICHD NIH HHS

Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is sustained by the production of new myofibers by means of the satellite cells. Survival of the satellite cells is a critical requirement for efficient muscle reconstitution. Necdin, a member of the MAGE proteins family, is expressed in satellite cell-derived myogenic precursors during perinatal growth and in the adult upon activation during muscle regeneration, where it plays an important role both in myoblast differentiation and survival. We show here that necdin exerts its pro-survival activity by counteracting the action of the pro-apoptotic protein Cell Cycle Apoptosis Regulatory Protein (CCAR1/CARP1) that we have identified as a new molecular interactor of necdin by two-hybrid screening. Necdin is responsible for the maintenance of CCAR1 protein levels, by implementing its ubiquitination and degradation through the proteasome. Taken together, these data shed new light on the molecular mechanism of necdin anti-apoptotic activity in myogenesis.

• Animals
• Apoptosis
• Apoptosis Regulatory Proteins/biosynthesis
• Cell Cycle Proteins/biosynthesis
• Cell Differentiation
• Cell Survival
• Gene Expression Regulation
• Gene Library
• Humans
• Mice
• Mice, Inbred C57BL
• Muscle Development
• Muscles/metabolism
• Myoblasts/cytology
• Nerve Tissue Proteins/biosynthesis
• Nuclear Proteins/biosynthesis
• Proteasome Endopeptidase Complex/metabolism
• Satellite Cells, Skeletal Muscle/cytology
• Two-Hybrid System Techniques
• Ubiquitin-Protein Ligases/biosynthesis

• type ii mage genes
• real-time pcr
• mouse embryonic carcinoma p19 cells
• neural differentiation
• retinoic acid induction

• Animals
• Cell Aggregation/drug effects
• Cell Differentiation/drug effects
• Cell Differentiation/genetics
• Cell Line, Tumor
• Cell Shape/drug effects
• Embryonal Carcinoma Stem Cells/metabolism
• Embryonal Carcinoma Stem Cells/pathology
• Gene Expression Regulation, Neoplastic/drug effects
• Genes, Neoplasm/genetics
• Melanoma-Specific Antigens/genetics
• Melanoma-Specific Antigens/metabolism
• Mice
• Neurons/drug effects
• Neurons/metabolism
• Neurons/pathology
• Real-Time Polymerase Chain Reaction/methods
• Reference Standards
• Reproducibility of Results
• Tretinoin/pharmacology

The finite replicative lifespan of cells, termed cellular senescence, has been proposed as a protective mechanism against the proliferation of oncogenically damaged cells, that fuel cancer. This concept is further supported by the induction of premature senescence, a process which is activated when an oncogene is expressed in normal primary cells as well as following intense genotoxic stresses. Thus, deregulation of genes that control this process, like the tumor suppressor p53, may contribute to promoting cancer by allowing cells to bypass senescence. A better understanding of the genes that contribute to the establishment of senescence is therefore warranted. Necdin interacts with p53 and is also a p53 target gene, although the importance of Necdin in the p53 response is not clearly understood.

In this study, we first investigated Necdin protein expression during replicative senescence and premature senescence induced by gamma irradiation and by the overexpression of oncogenic RasV12. Gain and loss of function experiments were used to evaluate the contribution of Necdin during the senescence process.

Necdin expression declined during replicative aging of IMR90 primary human fibroblasts or following induction of premature senescence. Decrease in Necdin expression seemed to be a consequence of the establishment of senescence since the depletion of Necdin in human cells did not induce a senescence-like growth arrest nor a flat morphology or SA-β-galactosidase activity normally associated with senescence. Similarly, overexpression of Necdin did not affect the life span of IMR90 cells. However, we demonstrate that in normal human cells, Necdin expression mimicked the effect of p53 inactivation by increasing radioresistance.

This result suggests that Necdin potentially attenuate p53 signaling in response to genotoxic stress in human cells and supports similar results describing an inhibitory function of Necdin over p53-dependent growth arrest in mice.

In vitro, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT), a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT) cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP) where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability.

• Animals
• Antigens, Polyomavirus Transforming/genetics
• Antigens, Polyomavirus Transforming/metabolism
• Apoptosis
• Biomarkers/metabolism
• Blotting, Northern
• Blotting, Western
• Cell Cycle
• Cell Proliferation
• Cell Transformation, Neoplastic/pathology
• Cells, Cultured
• Cystadenocarcinoma, Serous/genetics
• Cystadenocarcinoma, Serous/metabolism
• Female
• Fibroblasts/cytology
• Fibroblasts/metabolism
• Flow Cytometry
• Gene Expression Profiling
• Humans
• Immunoenzyme Techniques
• Mice
• NIH 3T3 Cells
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/metabolism
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Oligonucleotide Array Sequence Analysis
• Ovarian Neoplasms/genetics
• Ovarian Neoplasms/metabolism
• RNA, Messenger/genetics
• Real-Time Polymerase Chain Reaction
• Tumor Suppressor Protein p53/genetics
• Tumor Suppressor Protein p53/metabolism

• MOP-36056 Canadian Institutes of Health Research

White adipose tissues are composed mainly of white fat cells (adipocytes), which play a key role in energy storage and metabolism. White adipocytes are terminally differentiated postmitotic cells and arise from their progenitor cells (preadipocytes) or mesenchymal stem cells residing in white adipose tissues. Thus, white adipocyte number is most likely controlled by the rate of preadipocyte proliferation, which may contribute to the etiology of obesity. However, little is known about the molecular mechanisms that regulate preadipocyte proliferation during adipose tissue development. Necdin, which is expressed predominantly in postmitotic neurons, is a pleiotropic protein that possesses anti-mitotic and pro-survival activities. Here we show that necdin functions as an intrinsic regulator of white preadipocyte proliferation in developing adipose tissues. Necdin is expressed in early preadipocytes or mesenchymal stem cells residing in the stromal compartment of white adipose tissues in juvenile mice. Lentivirus-mediated knockdown of endogenous necdin expression in vivo in adipose tissues markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood. Furthermore, necdin-null mutant mice exhibit a greater expansion of adipose tissues due to adipocyte hyperplasia than wild-type mice when fed the high-fat diet during the juvenile and adult periods. Adipose stromal-vascular cells prepared from necdin-null mice differentiate in vitro into a significantly larger number of adipocytes in response to adipogenic inducers than those from wild-type mice. These results suggest that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development.

• Adipocytes/metabolism
• Adipocytes/physiology
• Animals
• Cell Differentiation/drug effects
• Cell Differentiation/genetics
• Cell Proliferation
• Cells, Cultured
• Down-Regulation/drug effects
• Down-Regulation/genetics
• Female
• Lentivirus/genetics
• Male
• Mice
• Mice, Inbred ICR
• Mice, Transgenic
• Nerve Tissue Proteins/antagonists & inhibitors
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/metabolism
• Nerve Tissue Proteins/physiology
• Nuclear Proteins/antagonists & inhibitors
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Nuclear Proteins/physiology
• RNA, Small Interfering/genetics
• RNA, Small Interfering/pharmacology
• Stem Cells/metabolism
• Stem Cells/physiology
• Stromal Cells/metabolism
• Stromal Cells/physiology
• Transfection

• Binding Sites
• Down-Regulation/genetics
• Down-Regulation/physiology
• Gene Expression Profiling
• Gene Expression Regulation, Neoplastic
• Genome, Human
• Humans
• Neoplasms/genetics
• Neoplasms/metabolism
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/physiology
• Nuclear Proteins/genetics
• Nuclear Proteins/physiology
• Oligonucleotide Array Sequence Analysis
• Promoter Regions, Genetic
• STAT3 Transcription Factor/genetics
• STAT3 Transcription Factor/physiology
• Transcription, Genetic

• P30 CA076292 NCI NIH HHS
• R01 CA115674 NCI NIH HHS
• R01-CA115674 NCI NIH HHS

• Adipocytes, Brown/cytology
• Adipogenesis
• Animals
• Cell Differentiation
• Cells, Cultured
• Cyclic AMP Response Element-Binding Protein/physiology
• Extracellular Signal-Regulated MAP Kinases/metabolism
• Forkhead Box Protein O1
• Forkhead Transcription Factors/physiology
• Insulin/physiology
• Insulin Receptor Substrate Proteins/physiology
• Insulin-Like Growth Factor I/physiology
• Mice
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/physiology
• Nuclear Proteins/genetics
• Nuclear Proteins/physiology
• Phosphorylation
• Promoter Regions, Genetic
• Signal Transduction

• DK081604 NIDDK NIH HHS
• DK046200 NIDDK NIH HHS
• P30 DK040561 NIDDK NIH HHS
• DK070722 NIDDK NIH HHS
• DK087317 NIDDK NIH HHS
• P30 DK46200 NIDDK NIH HHS
• RC1 DK087317 NIDDK NIH HHS
• P30 DK046200 NIDDK NIH HHS
• DK33201 NIDDK NIH HHS
• R21 DK070722 NIDDK NIH HHS
• R01 DK041430 NIDDK NIH HHS
• R01 DK077097 NIDDK NIH HHS
• K23 DK081604 NIDDK NIH HHS
• R01 DK033201 NIDDK NIH HHS
• KL2 RR025757 NCRR NIH HHS
• DK41430 NIDDK NIH HHS
• DK077097 NIDDK NIH HHS
• RR025757 NCRR NIH HHS

• Animals
• Apoptosis
• Embryo, Mammalian
• Female
• Gene Deletion
• Gene Expression Regulation
• Hindlimb/cytology
• Humans
• Lumbosacral Region/pathology
• Male
• Mice
• Mitosis/genetics
• Motor Activity/genetics
• Motor Neurons/cytology
• Motor Neurons/metabolism
• Motor Neurons/pathology
• Nerve Growth Factors/metabolism
• Nerve Tissue Proteins/deficiency
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/metabolism
• Nuclear Proteins/deficiency
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Prader-Willi Syndrome/metabolism
• Prader-Willi Syndrome/pathology
• Prader-Willi Syndrome/physiopathology
• Receptors, Tumor Necrosis Factor, Type I/metabolism
• Signal Transduction/genetics
• Spinal Cord/cytology
• Spinal Cord/pathology
• Tumor Necrosis Factor-alpha/metabolism

• cancer
• cancer/testis antigens
• cell cycle progression
• meiosis
• mitosis
• seminiferous epithelial cycle
• spermatids
• spermatocytes
• spermatogenesis
• spermatogonia
• spermatogonial stem cells
• spermatozoa
• testis
• tumorigenesis

• R01 HD056034 NICHD NIH HHS
• U54 HD029990 NICHD NIH HHS