Nek7 kinase targeting leads to early mortality, cytokinesis disturbance and polyploidy.

The mammalian NIMA-related kinases (Neks) are commonly referred to as mitotic kinases, although a definitive in vivo verification of this definition is largely missing. Reduction in the activity of Nek7 or its close paralog, Nek6, has previously been shown to arrest cells in mitosis, mainly at metaphase. In this study, we investigate the developmental and cellular roles of Nek7 kinase through the generation and analysis of Nek7-deficient mice. We show that absence of Nek7 leads to lethality in late embryogenesis or at early post-natal stages and to severe growth retardation. Mouse embryonic fibroblasts (MEFs) derived from Nek7(-/-) embryos show increase tendency for chromosomal lagging, micronuclei formation and cytokinesis failure. Tetraploidy and aneuploidy were commonly observed and their prevalence arises with MEFs passages. The frequency of multicentrosomal cells in the mutant's MEF cells was higher, and it commonly occurred concurrently with a binuclear phenotype, suggesting cytokinesis failure etiology. Lastly, the percentage of mutant MEF cells bearing primary cilia (PC) was low, whereas a cell population having two cilia appeared in the mutant MEFs. Taken together, these results confirm Nek7 as a regulator of cell division, and reveal it as an essential component for mammalian growth and survival. The intimate connection between tetraploidy, aneuploidy and cancer development suggests that Nek7 deregulation can induce oncogenesis.

A proline-rich motif in p53 is required for transactivation-independent growth arrest as induced by Gas1.

The involvement of p53 in regulating diverse cellular processes dictates that it must respond to multiple signaling mechanisms, thus coordinating the response to various "stress conditions." Genotoxic stress has served as a paradigm to dissect the transactivation-dependent branch of the pathway by which p53 can induce growth arrest. Alternate mechanisms have been invoked to explain transactivation-independent effects of p53, especially in the context of apoptosis. We have identified a p53-dependent pathway initiated by the gas1 product, a plasma membrane protein highly expressed during G0, which activates a transactivation-independent p53 growth arrest function. Through a detailed deletional analysis and site-specific mutagenesis of p53 we show that the Gas1-dependent signal transduction relies on a proline-rich region (amino acids 63-85) of murine p53. In vivo competition experiments using combinations of such mutants implicate this functional domain of p53 as a docking site in the transmission of antiproliferative signals.

Transgenic mouse model for studying the transcriptional activity of the p53 protein: age- and tissue-dependent changes in radiation-induced activation during embryogenesis.

The p53 tumor suppressor protein is a sequence-specific transcriptional activator of target genes. Exposure of cells to DNA damage results in accumulation of biochemically active p53, with consequent activation of p53-responsive promoters. In order to study how the transcriptional activity of the p53 protein is regulated in vivo, a transgenic mouse strain was generated. These mice harbor the p53-dependent promoter of the mdm2 gene, fused to a lacZ reporter gene. Induction of lacZ activity by DNA damage (ionizing radiation) was monitored in embryos of different p53 genotypes. The transgenic promoter was substantially activated in vivo following irradiation; activation required functional p53. The activation pattern became more restricted with increasing embryo age, as well as with the state of differentiation of a given tissue. Generally, maximal p53 activation occurred in rapidly proliferating, relatively less differentiated cells. A striking extent of haploinsufficiency was revealed-induction of promoter activity was far less efficient in mice carrying only one wild-type p53 allele. This suggests that normal levels of cellular p53 are limiting, and any further reduction already compromises the p53 response significantly. Thus, the activation potential of p53 is tightly controlled in vivo, both spatially and temporally, and an important element in this control is the presence of limiting basal levels of activatable p53.

Neu differentiation factor (Heregulin) activates a p53-dependent pathway in cancer cells.

Previously we reported that neu differentiation factor (NDF)/heregulin (HRG) elevates tyrosine phosphorylation of its receptors erbB-3, erbB-4, and erbB-2 (through heterodimer formation). We also showed that both NDF/HRG and antibodies to erbB-2 can arrest growth and induce differentiation in breast cancer cells. In this study, we report on the mechanism of NDF/HRG-induced cellular effects. We show that NDF/HRG and antibodies to erbB-2 receptors up-regulate expression of p53 by stabilizing the protein. This is accompanied by up-regulation of the p53 inducible gene, p21CIP1/WAF1, in a variety of cell lines: MCF7 and their derivatives (MCF7/HER2, MN1 and MCF-7-puro), ZR75T and LnCap cells. The induction of p21 is further enhanced when cells are treated with both NDF/HRG and DNA-damaging chemotherapeutic agents (i.e. doxorubicin). The NDF/HRG mediated induction of p21 is dependent on wildtype p53, as it fails to occur in cells expressing dominant negative p53 (MDD2). Furthermore, p21 induction is capable of inactivating cdk2 complexes as measured by Histone H1 phosphorylation assays. Finally, we show that in primary cultures of breast and other cancers, p21 is significantly induced in response to NDF/HRG treatment. Collectively, these observations suggest that the mechanism of breast cancer cell growth inhibition and differentiation via erbB receptors activation is through a p53-mediated pathway.

E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis.

The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G0 and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors.

Loss of p53 function through PAX-mediated transcriptional repression.

Direct interactions between the genes that regulate development and those which regulate the cell cycle would provide a mechanism by which numerous biological events could be better understood. We have identified a direct role for PAX5 in the control of p53 transcription. In primary human diffuse astrocytomas, PAX5 expression inversely correlated with p53 expression. The human p53 gene harbours a PAX binding site within its untranslated first exon that is conserved throughout evolution. PAX5 and its paralogues PAX2 and PAX8 are capable of inhibiting both the p53 promoter and transactivation of a p53-responsive reporter in cell culture. Mutation of the identified binding site eliminates PAX protein binding in vitro and renders the promoter inactive in cells. These data suggest that PAX proteins might regulate p53 expression during development and propose a novel alternative mechanism for tumour initiation or progression, by which loss of p53 function occurs at the transcriptional level.

Biochemical properties and biological effects of p53.

Over the past year, insights have been made into the biochemistry and biological effects of p53. The high-resolution three-dimensional structure has been determined for the central core and carboxy-terminal domain of the protein, important p53 target genes (such as WAF1) have been identified, and insight has been gained into the relationship between p53-mediated growth arrest and apoptosis.

Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal.

Overexpression of wild-type p53 in p53-deficient leukemic cells induces apoptosis, which can be inhibited by hematopoietic survival factors. This suggests that p53 may contribute to survival factor dependence. To assess the role of wild-type p53 in mediating apoptosis following survival factor withdrawal, we interfered with endogenous p53 activity in interleukin-3 (IL-3)-dependent cells. Extended survival without IL-3 was conferred by recombinant retroviruses encoding either a full-length p53 mutant or a C-terminal p53 miniprotein, both of which can act as negative-dominant inhibitors of wild-type p53. On the other hand, excess wild-type p53 activity failed to elicit apoptosis as long as IL-3 was present. We propose that p53 is a positive, though not exclusive, mediator of survival factor dependence in hematopoietic cells.

A role for CdxA in gut closure and intestinal epithelia differentiation.

CdxA is a homeobox gene of the caudal type that was previously shown to be expressed in the endoderm-derived gut epithelium during early embryogenesis. Expression of the CDXA protein was studied during intestine morphogenesis from stage 11 (13 somites) to adulthood in the chicken. The CDXA protein can be detected during all stages of gut closure, from stage 11 to 5 days of incubation, and is mainly localized to the intestinal portals, the region where the splanchnopleure is undergoing closure. In this region, which represents the transition between the open and closed gut, the CDXA protein is restricted to the endoderm-derived epithelium. At about day 5 of incubation, the process of formation of the previllous ridges begins, which marks the beginning of the morphogenesis of the villi. From this stage to day 11 expression of CDXA is localized to the epithelial lining of the intestine. In parallel, a gradual increase in CDXA protein expression begins in the mesenchyme that is close in proximity to the CDXA-positive endoderm. Maximal CDXA levels in the mesenchyme are observed at day 9 of incubation. During days 10 and 11 CDXA levels in the mesenchyme remain constant, and by day 12 CDXA becomes undetectable in these cells and the epithelium again becomes the main site of expression. From day 12 of incubation until adulthood the CDXA protein is present in the intestinal epithelium. Until day 18 of incubation expression can be detected along the whole length of the villus with a stronger signal at the tip. With hatching the distribution along the villi changes so that the main site of CDXA protein expression is at the base of the villi and in the crypts. The transient expression of CDXA in the mesenchyme between days 5 and 11 may be related to the interactions taking place between the mesenchyme and the epithelium that ultimately result in the axial specification of the alimentary canal and the differentiation of its various epithelia. The main CDXA spatial distribution during morphogenesis suggests a tight linkage to the formation and differentiation of the intestinal epithelium itself. CDXA appears to play a role in the morphogenetic events leading to closure of the alimentary canal. During previllous ridge formation the CDXA protein is transiently expressed in the mesenchymal cells thought to provide instructive interactions for the regionalization and differentiation of the gut epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

The chicken CdxA homeobox gene and axial positioning during gastrulation.

The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation. Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak. The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast. In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E. coli and generated monoclonal antibodies against it. Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies. Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein. Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression. The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages. As the primitive streak elongates, the CdxA stripe of expression moves anteriorly. By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak. CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo. These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.

mdm2 expression is induced by wild type p53 activity.

We have recently characterized a 95 kDa protein, p95, which exhibits enhanced binding to temperature-sensitive p53 (ts-p53) when cells are shifted down to 32.5 degrees C, a temperature at which ts-p53 possesses wild-type (wt)-like activities. In the present study we show that p95 is a product of the mdm2 putative proto-oncogene. The enhanced complex formation of mdm2 with ts-p53 in cells maintained at 32.5 degrees C is due to an elevation in total mdm2 protein levels following the temperature shift. We further demonstrate that the induction of mdm2 expression by t p53 activity is at the mRNA level. The induction occurs with very rapid kinetics and does not require de novo protein synthesis, suggesting a direct involvement of p53 in the process. Based on these data and on recent findings implicating p53 as a transcription factor, we suggest that the mdm2 gene is a target for activation by wt p53. In view of the ability of mdm2 to act as a specific antagonist of p53 activity, this induction process may serve to tightly autoregulate p53 activity in living cells.