RB1
Retinoblastoma-associated protein (p105-Rb) (p110-RB1) (pRb) (Rb) (pp110)
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Nodular fasciitis (NF) is a self-limited tumorous lesion occurring in the upper as well as lower extremities. NF is composed of a proliferation of "primary culture"-like myofibroblastic cells with nuclear atypia and large nucleoli, thus mimicking sarcoma. NF harbors a promoter-swapping fusion gene containing the entire coding region of [i]USP6[/i] gene. Therefore, NF is a tumor with a fusion oncogene but self-limited. In order to explore why NF is self-limited, we examined whether myofibroblastic cells in NF express p16 protein, a gene product of [i]CDKN2A[/i] gene and an inhibitor of cyclin-dependent kinase 4 (CDK4) as well as one of the hallmarks of cellular senescence. We immunohistochemically demonstrated strong and diffuse expression of p16 in myofibroblastic cells in 11 out of 15 cases of NF, and strong but partial expression in the remaining 4 of the cases. We also showed that 15 out of 15 cases of NF were immunohistochemically negative or only showed focal and faint immunopositivity for CDK4, murine double minute 2 (MDM2), and TP53 proteins. Furthermore, there were no significant changes in the copy number of [i]CDKN2A, CDK4[/i] and [i]MDM2[/i] genes, and no significant mutations in [i]TP53, RB1,[/i] and [i]CDKN2A[/i] genes in 1 case of NF selected. These data suggest a possible involvement in cell cycle arrest and presumed cellular senescence by p16 in myofibroblastic cells in NF. This may explain the self-limited as well as inflammatory nature of NF as a senescence-associated secretory phenotype.
Keywords
- CDK4
- MDM2
- TP53
- nodular fasciitis
- p16
- senescence
Senescent cells have lost their capacity for proliferation and manifest as irreversibly in cell cycle arrest. Many membrane receptors, including G protein-coupled receptors (GPCRs), initiate a variety of intracellular signaling cascades modulating cell division and potentially play roles in triggering cellular senescence response. GPCR kinases (GRKs) belong to a family of serine/threonine kinases. Although their role in homologous desensitization of activated GPCRs is well established, the involvement of the kinases in cell proliferation is still largely unknown. In this study, we isolated GRK4-GFP expressing HEK293 cells by fluorescence-activated cell sorting (FACS) and found that the ectopic expression of GRK4 halted cell proliferation. Cells expressing GRK4 (GRK4( )) demonstrated cell cycle G1/G0 phase arrest, accompanied with significant increase of senescence-associated-β-galactosidase (SA-β-Gal) activity. Expression profiling analysis of 78 senescence-related genes by qRT-PCR showed a total of 17 genes significantly changed in GRK4( ) cells (≥ 2 fold, p < 0.05). Among these, 9 genes - AKT1, p16 , p27 , p19 , IGFBP3, MAPK14, PLAU, THBS1, TP73 - were up-regulated, while 8 genes, Cyclin A2, Cyclin D1, CDK2, CDK6, ETS1, NBN, RB1, SIRT1, were down-regulated. The increase in cyclin-dependent kinase inhibitors (p16, p27) and p38 MAPK proteins (MAPK14) was validated by immunoblotting. Neither p53 nor p21 protein was detectable, suggesting no p53 activation in the HEK293 cells. These results unveil a novel function of GRK4 on triggering a p53-independent cellular senescence, which involves an intricate signaling network.
MeSH Terms
- Cell Division
- Cell Line, Tumor
- Cell Proliferation
- Cellular Senescence
- Flow Cytometry
- G-Protein-Coupled Receptor Kinase 4
- Gene Expression Profiling
- Gene Expression Regulation
- HEK293 Cells
- Humans
- MCF-7 Cells
- Transcriptome
- Tumor Suppressor Protein p53
Keywords
- Cellular senescence
- G protein-coupled receptor kinase 4
- Gene expression profiling
- p53-independent senescence
Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors. MSC cultures contain subpopulations of mesenchymal stem cells and committed progenitors that can differentiate into mesodermal derivatives: adipocytes, chondrocytes, and osteocytes. These stem cells and committed osteoblast precursors are the cell of origin in osteosarcoma, and RB1 gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated RB1 do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated RB1 the radiation treatment did not increase apoptosis, necrosis or senescence versus untreated cells. Following radiation, CFU analysis showed a discrete number of cells with clonogenic capacity in cultures with silenced RB1. We extended our analysis to the other members of retinoblastoma gene family: RB2/P130 and P107. Also in the MSCs with silenced RB2/P130 and P107 we detected the presence of cells with unrepaired DNA following X-ray irradiation. Cells with unrepaired DNA may represent a reservoir of cells that may undergo neoplastic transformation. Our study suggests that, following radiotherapy, cancer patients with mutations of retinoblastoma genes may be under strict controls to evaluate onset of secondary neoplasms following radiotherapy.
MeSH Terms
- Apoptosis
- Cell Cycle
- Cell Lineage
- Cell Survival
- Cell Transformation, Neoplastic
- Cellular Senescence
- Colony-Forming Units Assay
- DNA Damage
- DNA Repair
- Gene Silencing
- Histones
- Humans
- Mesenchymal Stem Cells
- Radiotherapy
- Retinoblastoma Protein
- Retinoblastoma-Like Protein p107
- X-Rays
Keywords
- Apoptosis
- DNA damage
- mesenchymal stem cells
- senescence
Cellular senescence impedes cancer progression by limiting uncontrolled cell proliferation. To identify new genetic events controlling senescence, we performed a small interfering RNA screening human cancer cells and identified a number of targets potentially involved in senescence of MDA-MB-231 human breast cancer cells. Importantly, we showed that knockdown of RAD21 resulted in the appearance of several senescent markers, including enhanced senescence-associated β-galactosidase activity and heterochromatin focus formation, as well as elevated p21 protein levels and RB1 pathway activation. Further biochemical analyses revealed that RAD21 knockdown led to the downregulation of c-Myc and its targets, including CDK4, a negative regulator of RB1, and blockedRB1 phosphorylation (pRB1), and the RB1-mediated transcriptional repression of E2F. Moreover, c-Myc downregulation was partially mediated by proteasome-dependent degradation within promyelocytic leukemia (PML) nuclear bodies, which were found to be highly abundant during RAD21 knockdown-induced senescence. Exogenous c-Myc reconstitution rescued cells from RAD21 silencing-induced senescence. Altogether, data arising from this study implicate a novel function of RAD21 in cellular senescence in MDA-MB-231 cells that is mainly dependent onRB1 pathway activation via c-Myc downregulation.
MeSH Terms
- Breast Neoplasms
- Cell Cycle Proteins
- Cell Line, Tumor
- Cellular Senescence
- DNA-Binding Proteins
- Down-Regulation
- Female
- Gene Expression Regulation, Neoplastic
- Gene Knockdown Techniques
- Humans
- Nuclear Proteins
- Phosphoproteins
- Proto-Oncogene Proteins c-myc
- RNA, Small Interfering
- Retinoblastoma Binding Proteins
- Signal Transduction
- Ubiquitin-Protein Ligases
Keywords
- MDA-MB-231
- RAD21
- RB1
- SENESCENCE
- c-Myc
The mechanisms by which retinoblastoma 1 (RB1) mediates oncosuppressive functions are still being elucidated. We found that radiation-induced senescence in the bone depends on RB1 and is associated with the secretion of multiple bioactive factors, including interleukin-6 (IL-6), as well as with the infiltration of natural killer T (NKT) cells. Importantly, the inhibition of RB1, IL-6 or NKT cells predisposed mice to radiation-induced osteosarcomas, unveiling a cancer cell-extrinsic mechanisms that underlie the oncosuppressive activity of RB1.
Keywords
- interleukin-6
- natural killer T cells
- radiation
- retinoblastoma 1
- senescence
- senescence-associated secretory phenotype (SASP)
Autophagy-related 1 (Atg1)/Unc-51-like protein kinases (ULKs) are evolutionarily conserved proteins that play critical physiological roles in controlling autophagy, cell growth and neurodevelopment. RB1-inducible coiled-coil 1 (RB1CC1), also known as PTK2/FAK family-interacting protein of 200 kDa (FIP200) is a recently discovered binding partner of ULK1. Here we isolated the Drosophila RB1CC1/FIP200 homolog (Fip200/CG1347) and showed that it mediates Atg1-induced autophagy as a genetically downstream component in diverse physiological contexts. Fip200 loss-of-function mutants experienced severe mobility loss associated with neuronal autophagy defects and neurodegeneration. The Fip200 mutants were also devoid of both developmental and starvation-induced autophagy in salivary gland and fat body, while having no defects in axonal transport and projection in developing neurons. Interestingly, moderate downregulation of Fip200 accelerated both developmental growth and aging, accompanied by target of rapamycin (Tor) signaling upregulation. These results suggest that Fip200 is a critical downstream component of Atg1 and specifically mediates Atg1's autophagy-, aging- and growth-regulating functions.
MeSH Terms
- Aging
- Animals
- Autophagy
- Autophagy-Related Protein-1 Homolog
- Axons
- Brain
- Conserved Sequence
- Drosophila Proteins
- Drosophila melanogaster
- Homeostasis
- Motor Activity
- Mutation
- Nerve Degeneration
- Protein-Serine-Threonine Kinases
- Ribosomal Protein S6 Kinases
- Signal Transduction
- TOR Serine-Threonine Kinases
- Time Factors
- Ubiquitin
- Vacuoles
Keywords
- Drosophila
- aging
- autophagy
- growth
- neurodegeneration