NIPA Deficiency Leads To Acceleration Of The Lymphoma Development In EµMyc Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3681-3681
Author(s):  
Anna Lena Illert ◽  
Cathrin Klingeberg ◽  
Corinna Albers ◽  
Stephan w. Morris ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Conflicts Of Interest ◽  
Control Cell ◽  
Cell Cycle Checkpoint ◽  
Conditional Knockout ◽  
Substantial Contribution ◽  
In Vivo Model ◽  
Checkpoint Control ◽  
Focus Formation ◽  
The Impact

Abstract Timely degradation of proteins that control cell proliferation and apoptosis is an essential mechanism in keeping normal growth from turning into runaway malignancy. We previously reported the cloning of NIPA (Nuclear-Interaction-Partner-of-ALK) and characterized it as a F-Box-protein that defines an oscillating E3-ubiquitin-ligase. Using a conditional-knockout strategy we inactivated Nipa and found Nipa-/- animals to be viable, but sterile due to a block of spermatogenesis. Our studies demonstrate that loss of Nipa has no substantive effect on physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, Nipa checkpoint control can be unmasked by oncogenic c-Myc-transformation. Here we show significant differences in c-Myc-induced transformation: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced. Moreover, Nipa-deficiency leads to premature senescence in cultured primary MEFs. Ectopic reexpression of Nipa resulted vice versa in delayed senescence of knockout MEFs. Next, we sought to know, whether increased apoptosis in Nipa-/- c-Myc-transduced MEFs is dependent on a functional p53-Axis. Interestingly, the effect of Nipa deficiency on c-Myc-mediated transformation was totally abolished by p53-knockdown. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53, suggesting the importance of p53 in Nipa-induced cell death. Looking in more detail on the c-myc-p53 axis we detected a substantial increase in Arf-p19 levels in Nipa-/- cells. Moreover, Nipa-knockdown in Zn-inducible-Arf-NIH/3T3 cells lead to stabilization of Arf p19. To test the impact of these findings in a relevant in-vivo model we intercrossed Nipa-/- animals with a transgene EµMyc-Strain. Nipa-/-EµMycTG/wt animals developed lymphomas within a significantly shorter latency than Nipa+/+EµMycTG/wt animals. Furthermore, lymphomas of knockout animals were more aggressive. FACS- and biochemical-analyses showed no gross differences between Nipa-/- and wt lymphomas except highly elevated Arf-p19 levels in Nipa-/- lymphomas, pointing to an important role of Nipa in Myc-p19-signalling. Taken together our results highlight the functional importance of the Nipa-p53-axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis. Disclosures: No relevant conflicts of interest to declare.

NIPA Checkpoint Control In Oncogenic Transformation Is Dependent on p53

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4178-4178
Author(s):  
Anna Lena Illert ◽  
Hiroyuki Kawaguchi ◽  
Corinna Albers ◽  
Melanie Sickinger ◽  
Ulrich Keller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin B1 ◽  
Conditional Knockout ◽  
Substantial Contribution ◽  
Dependent Manner ◽  
Checkpoint Control ◽  
Oncogenic Transformation ◽  
Wild Type ◽  
Focus Formation ◽  
Wild Type Mefs

Abstract Abstract 4178 The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding NIPA. NIPA-deficient animals are viable, but sterile due to a block of spermatogenesis. Moreover, our studies demonstrate that loss of NIPA has no substantive effect on the physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interestingly, NIPA checkpoint control can be unmasked by oncogenic transformation by c-Myc. Here we show that transformed focus formation assays revealed highly significant differences in c-Myc-induced transformation in NIPA-deficient and wild-type MEFs. c-Myc transduction caused a pronounced upregulation of cyclin-B in NIPA-null MEFs, which was completely reversible by ectopic NIPA expression. The increased cyclin-B1 expression after c-Myc transduction in the absence of NIPA has considerable functional consequences for the cells: Focus formation ability of c-Myc-infected Nipa-/- MEFs was greatly reduced in comparison to wild-type MEFs (24.6% vs. 100%). Moreover, c-Myc expression caused 12.8% apoptotic subG1 cells in wild-type MEFs, whereas Nipa-/- MEFs were more affected by c-Myc-induced apoptosis (22.45%). Next, we sought to know, whether increased apoptosis in Nipa-deficient c-Myc transduced MEFs is dependent on a functional p53-Axis. Therefore, Nipa-wildtype and knockout MEFs were first infected with a retroviral Supernatant encoding for a p53Mir- and thereafter with the oncogene c-Myc. Interestingly the effect of Nipa knockout on c-Myc-mediated oncogenic transformation was totally abolished by the knock-down of p53. We observed no differences in focus formation ability or growth behaviour in Nipa-/- MEFs with inactivated p53 in comparison to wildtype cells, suggesting the importance of functional p53 in Nipa-induced cell death. Taken together, our data demonstrate that NIPA is required for efficient c-Myc transformation in a p53-dependent manner. Moreover, our results highlight the functional importance of the NIPA-p53 axis in cell cycle regulation and suggest that deregulation of the protein provides a substantial contribution during the process of tumorigenesis. Disclosures: No relevant conflicts of interest to declare.

NIPA Phosphorylation and Inactivation at G2/M Is Mediated by ERK2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2513-2513
Author(s):  
Michael Zech ◽  
Anna Lena Illert ◽  
Corinna Albers ◽  
Florian Bassermann ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
High Efficiency ◽  
Conflicts Of Interest ◽  
Cyclin B1 ◽  
Cell Cycle Checkpoint ◽  
Critical Event ◽  
Checkpoint Control ◽  
Cycle Progression

Abstract Abstract 2513 Poster Board II-490 Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, contributing to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3-ligase. The SCF-NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1, a critical event for proper G2/M transition. Thus, SCF-NIPA executes an important G2/M checkpoint control. We recently specified three serine residues Ser 354, 359 and 395 implicated in NIPA phosphorylation at G2/M. These data suggest a sequential NIPA phosphorylation, where initial Ser 354 and 359 phosphorylation is most crucial for SCF-NIPA inactivation by dissociating the SCF-NIPA complex. Here we aimed to find the kinase responsible in this initial most important phosphorylation step. Using in vitro kinase assays we identified both ERK1 and ERK2 to phosphorylate NIPA with high efficiency. Mutation of either Ser 354 or Ser 359 abolished ERK-dependent NIPA phosphorylation. Inhibition of ERK1/2 activity in cell lines by specific inhibitors resulted in decreased NIPA phosphorylation at G2/M. To differentiate between phosphorylation by ERK1 and ERK2, we combined cell cycle analysis with stable expression of microRNA's targeting both isoforms. To this end NIH/3T3 cells were retrovirally transduced with microRNAs targeting ERK1 and 2 and cell cycle progression was analysed by BRDU/PI labeling. Using this approach, we are able to show that ERK2 but not ERK1 mediates NIPA phosphorylation at G2/M. Furthermore, ERK2 silencing leads to a distinct phenotype in cell cycle progression with a delay of ERK2 knockdown cells at the G2/M transition. Thus, our data indicate, that the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation could be in part due to the differential ability of these kinases to phosphorylate and inactivate NIPA at G2/M. Since checkpoint proteins such as NIPA are constitutively inactivated in tumor cells ERK2 might represent an interesting target to reconstitute important cell cycle checkpoint controls in malignant cells. Disclosures: No relevant conflicts of interest to declare.

DNA damage checkpoint kinases in cancer

2020 ◽  
Vol 22 ◽  
Author(s):  
Hannah L. Smith ◽  
Harriet Southgate ◽  
Deborah A. Tweddle ◽  
Nicola J. Curtin
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Environmental Dna ◽  
Cell Cycle Checkpoint ◽  
Checkpoint Control ◽  
Integrated Network ◽  
Checkpoint Kinases ◽  
G1 Checkpoint ◽  
Cycle Checkpoint ◽  
High Level

Abstract DNA damage response (DDR) pathway prevents high level endogenous and environmental DNA damage being replicated and passed on to the next generation of cells via an orchestrated and integrated network of cell cycle checkpoint signalling and DNA repair pathways. Depending on the type of damage, and where in the cell cycle it occurs different pathways are involved, with the ATM-CHK2-p53 pathway controlling the G1 checkpoint or ATR-CHK1-Wee1 pathway controlling the S and G2/M checkpoints. Loss of G1 checkpoint control is common in cancer through TP53, ATM mutations, Rb loss or cyclin E overexpression, providing a stronger rationale for targeting the S/G2 checkpoints. This review will focus on the ATM-CHK2-p53-p21 pathway and the ATR-CHK1-WEE1 pathway and ongoing efforts to target these pathways for patient benefit.

RhoA Is An Essential Regulator of Mitosis and Survival During Hematopoietic Stem Cell Differentiation to Multipotent Progenitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1273-1273
Author(s):  
Xuan Zhou ◽  
Jaime Meléndez ◽  
Yuxin Feng ◽  
Richard Lang ◽  
Yi Zheng
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Stem Cell Differentiation ◽  
Conditional Knockout ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
Multipotent Progenitors

Abstract Abstract 1273 The maintenance and differentiation of hematopoietic stem cells (HSC) are critical for blood cell homeostasis, which is tightly regulated by a variety of factors. In spite of extensive investigation of HSC biology, however, the mechanism of regulation of HSC and progenitor cell division, particularly the unique molecular events controlling the mitosis process during HSC differentiation, remains unclear. RhoA GTPase is a critical intracellular signaling nodal that has been implicated in signal transduction from cytokines, chemokines, wnt/notch/shh, and adhesion molecules to impact on cell adhesion, migration, cell cycle progression, survival and gene expression. Recent mouse genetic studies in keratinocytes and embryonic fibroblast cells showed that RhoA is a key regulator of mitosis. By using an interferon-inducible RhoA conditional knockout mouse model (Mx-cre;RhoAlox/lox), we have made the discovery that RhoA plays an indispensible role in primitive hematopoietic progenitor differentiation through the regulation of mitosis and survival. RhoA deficient mice die at ∼10 days because of hematopoietic failure, as evidenced by a loss of bone marrow, splenocyte and PB blood cells. Syngenic as well as reverse transplant experiments demonstrate that these effects are intrinsic to the hematopoietic compartment. RhoA loss results in pancytopenia associated with a rapid exhaustion of the lin−c-kit+ (LK) phenotypic progenitor population (within 4 days after two polyI:C injections). Meanwhile, the lin−c-kit+sca1+ (LSK) primitive cell compartment is transiently increased in BM after RhoA deletion due to a compensatory loss of quiescence and increased cell cycle. Interestingly, we find that within the LSK population, there is a significant accumulation of LSKCD34+Flt2− short-term HSCs (ST-HSC) and a corresponding decrease in frequency of LSKCD34+Flt2+ multipotent progenitors (MPPs). Consistent with these phenotypes, the LK and more differentiated hematopoietic cell populations of RhoA knockout mice show an increased apoptosis while the survival activities of LSK and more primitive compartments of WT and RhoA KO mice remain comparable. These data suggest that RhoA plays an indispensible role in the step of ST-HSCs differentiation to MPP cells, possibly through the regulation of MPP cell survival. This hypothesis is further supported by a competitive transplantation experiment. Deletion of RhoA in a competitive transplantation model causes an extinction of donor derived (CD45.2+) differentiated cells (myeloid, erythroid, T and B cells) in the peripheral blood. Interestingly, bone marrow CD45.2+ LSK cells are only marginally affected by deletion of RhoA and RhoA−/− LSK cells are able to engraft into 2nd recipient, whereas CD45.2+ LK and more differentiated cells are mostly eliminated after RhoA deletion. This effect is associated with a decrease in the survival of CD45.2+ RhoA−/− LK, but not LSK cells. Further in vitro culture of isolated lin− progenitors demonstrates that RhoA deficiency results in a failure of cytokinesis, causing an accumulation of multinucleated cells, further suggesting that RhoA is essential for the cytokinesis of hematopoietic progenitors. Surprisingly, the well-defined Rho downstream target, actomyosin machinery, does not appear to be affected by RhoA knockout. We are further exploring the mechanism of RhoA contribution to the differentiation of HSCs by dissecting the signaling and functional relationship of RhoA regulated survival activity and cell cycle mitosis in early hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

Loss Of NIPA Leads To Defects In The Repair Of DNA Double Strand Breaks In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2488-2488
Author(s):  
Anna Lena Illert ◽  
Cristina Antinozzi ◽  
Hiroyuki Kawaguchi ◽  
Michal Kulinski ◽  
Christine Klitzing ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Meiotic Prophase ◽  
Conflicts Of Interest ◽  
Cyclin B1 ◽  
Double Strand Breaks ◽  
Conditional Knockout ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Chromosome Axis

Abstract Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding Nipa. Nipa-deficient animals are viable, but show a lower birth rate and a reduced body weight. Furthermore, Nipa-deficient males were sterile due to a block of spermatogenesis during meiotic prophase. Virtually no spermatocytes progress beyond a late-zygotene to early-pachytene stage and reach an aberrant stage, with synaptonemal complex disassembly and incomplete synapsis. Nipa-/- females are sub-fertile with an early and severe meiotic defect during embryogenesis with extensive apoptosis in early prophase (E13.5-E14.5). Here we report, that Nipa-/- meiocytes exhibit persistent cytological markers for DNA double strand break repair proteins (like DMC1, RAD51) in meiotic prophase with more than twice as many DMC1 foci as control animals. Kinetic analysis of the first wave of spermatogenesis showed increased DMC1/RAD51 foci in Nipa-/- cells as soon as early-pachynema cells appear (13-14 days post partum). Moreover, we show that Nipa deficiency does not lead to a defect in meiotic sex chromosome inactivation despite epithelial stage IV apoptosis. Nipa-deficient spermatocytes exhibit numerous abnormalities in staining of chromosome axis associated proteins (like SYCP3 and STAG3) indicating that chromosome axis defects were associated with compromised chromosome axis integrity leading to overt chromosome fragmentation. Further in vitro analyses with bleomycin treated MEFs displayed high pH2AX levels in cells lacking NIPA. Repair of DNA DSB seemed to be abolished in these cells as the pH2AX-level were sustained and still visible after 90 min of timecourse, where wildtype cells already repaired sides of DNA Damage. Consistent with these findings NIPA-deficient spleen cells showed compromised DNA Damage repair measured in a comet assay with a significantly longer olive tail moment in NIPA knockout cells under repair conditions. Taken together, the phenotype of Nipa-knockout mice is a definitive proof of the meiotic significance of NIPA and our results show a new, unsuspected role of NIPA in chromosome stability and the repair of DNA double strand breaks. Disclosures: No relevant conflicts of interest to declare.

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