Kazushige Furuya

UFD2a mediates the proteasomal turnover of p73 without promoting p73 ubiquitination

p73 protein level is kept extremely low in mammalian cultured cells and its stability may be regulated by not only the ubiquitin/proteasome-dependent proteolysis but also through other unidentified mechanisms. Here, we found for the first time that p73 is physically as well as functionally associated with the U-box-type E3/E4 ubiquitin ligase UFD2a. The immunoprecipitation experiments demonstrated that this interaction is mediated by the COOH-terminal region of p73α containing SAM domain. During the cisplatin-induced apoptosis in SH-SY5Y neuroblastoma cells, p73α accumulated at a protein level, whereas the endogenous UFD2a was significantly reduced in response to cisplatin. Ectopic expression of UFD2a decreased the half-life of p73α in association with a significant inhibition of the p73α-mediated transactivation as well as proapoptotic activity. Downregulation of endogenous UFD2a by antisense strategy resulted in a remarkable accumulation of p73α. Unexpectedly, UFD2a-mediated degradation of p73α was sensitive to the proteasomal inhibitor, however, UFD2a did not affect the ubiquitination levels of p73α. Taken together, our present findings imply that UFD2a might promote the proteasomal turnover of p73 in a ubiquitination-independent manner, and also suggest that UFD2a might play an important role in the regulation of cisplatin-induced apoptosis mediated by p73.

ATM-dependent nuclear accumulation of IKK- α plays an important role in the regulation of p73-mediated apoptosis in response to cisplatin

I kappa B kinase (IKK) complex plays an important role in the regulation of signaling pathway that activates nuclear factor–kappa-B (NF-κB). Recently, we reported that cisplatin (CDDP) treatment causes a remarkable nuclear accumulation of IKK-α in association with stabilization and activation of p73. However, underlying mechanisms of CDDP-induced nuclear accumulation of IKK-α are elusive. Here, we found that ataxia–telangiectasia mutated (ATM) is one of upstream mediators of IKK-α during CDDP-induced apoptosis. In response to CDDP, ATM was phosphorylated at Ser-1981, which was accompanied with nuclear accumulation of IKK-α in HepG2 cells, whereas CDDP treatment had undetectable effects on IKK-α in ATM-deficient cells. Indirect immunofluorescence experiments demonstrated that phosphorylated form of ATM colocalizes with nuclear IKK-α in response to CDDP. In vitro kinase assay indicated that ATM phosphorylates IKK-α at Ser-473. Moreover, IKK-α-deficient MEFs displayed CDDP-resistant phenotype as compared with wild-type MEFs. Taken together, our present results suggest that ATM-mediated phosphorylation of nuclear IKK-α, which stabilizes p73, is one of the main apoptotic pathways in response to CDDP.

NFBD1/MDC1 associates with p53 and regulates its function at the crossroad between cell survival and death in response to DNA damage.

NFBD1/MDC1, which belongs to the BRCT superfamily, has an anti-apoptotic activity and contributes to the early cellular responses to DNA damage. Here we found that NFBD1 protects cells from apoptotic cell death by inhibiting phosphorylation of p53 at Ser-15 under steady state as well as early phase of DNA damage, thereby blocking its transcriptional and pro-apoptotic activities. During late phase of DNA damage, a remarkable reduction of NFBD1 was observed in dying but not in surviving A549 cells bearing wild-type p53. Small interference RNA-mediated knockdown of the endogenous NFBD1 resulted in an increase in sensitivity to adriamycin in A549 cells but not in p53-deficient H1299 cells. Immunoprecipitation and luciferase reporter analyses demonstrated that NFBD1 binds to the NH2-terminal region of p53 and strongly inhibits its transcriptional activity. Additionally, BRCT domains, which can interact with p53, reduced the adriamycin-induced phosphorylation levels of p53 at Ser-15 and also suppressed the transcriptional activity of p53. Thus, our present findings strongly suggest that NFBD1 plays an important role in the decision of cell survival and death after DNA damage through the regulation of p53.

Stabilization of p73 by nuclear IκB kinase-α mediates cisplatin-induced apoptosis

In response to DNA damage, p53 and its homolog p73 have a function antagonistic to NF-κB in deciding cell fate. Here, we show for the first time that p73, but not p53, is stabilized by physical interaction with nuclear IκB kinase (IKK)-α to enhance cisplatin (CDDP)-induced apoptosis. CDDP caused a significant increase in the amounts of nuclear IKK-α and p73α in human osteosarcoma-derived U2OS cells. Ectopic expression of IKK-α prolonged the half-life of p73 by inhibiting its ubiquitination and thereby enhancing its transactivation and pro-apoptotic activities. Consistent with these results, small interfering RNA-mediated knockdown of endogenous IKK-α inhibited the CDDP-mediated accumulation of p73α. The kinase-deficient mutant form of IKK-α interacted with p73α, but failed to stabilize it. Furthermore, CDDP-mediated accumulation of endogenous p73α was not detected in mouse embryonic fibroblasts (MEFs) prepared from IKK-α-deficient mice, and CDDP sensitivity was significantly decreased in IKK-α-deficient MEFs compared with wild-type MEFs. Thus, our results strongly suggest that the nuclear IKK-α-mediated accumulation of p73α is one of the novel molecular mechanisms to induce apoptotic cell death in response to CDDP, which may be particularly important in killing tumor cells with p53 mutation.

Identification of protein kinase A catalytic subunit β as a novel binding partner of p73 and regulation of p73 function

Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit β (PKA-Cβ) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73α associated with PKA-Cβ in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73α. In contrast, p53 failed to bind to PKA-Cβ. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73α-(1–130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cβ resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73α, whereas a kinase-deficient mutant of PKA-Cβ had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73α. Of particular interest, PKA-Cβ facilitated the intramolecular interaction of p73α, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cβ-mediated inhibitory mechanism of p73 function.

NF-κB regulates the stability and activity of p73 by inducing its proteolytic degradation through a ubiquitin-dependent proteasome pathway

Nuclear factor kappa B (NF-κB), which exists as heterodimeric complexes composed of p50 and p65, has been shown to play an important role in cell survival processes. In the present study, we found for the first time that NF-κB has an ability to induce the ubiquitin-dependent proteasomal degradation of proapoptotic p73α. The activation of NF-κB in tumor necrosis factor α (TNF-α)-stimulated H1299 cells resulted in a significant reduction in the amounts of the endogenous p73α. Consistent with these results, TNF-α-mediated downregulation of p73α was observed in wild-type (WT) mouse embryonic fibroblasts (MEFs) but not in p65-deficient MEFs. Ectopic expression of NF-κB decreased a half-life of p73α by increasing its ubiquitination levels, and thereby inhibiting the transcriptional activity as well as proapoptotic function of p73α, whereas NF-κB had undetectable effects on p53. Immunoprecipitation experiments demonstrated that, under our experimental conditions, NF-κB does not bind to p73α in mammalian cultured cells. In contrast to WT p65, the COOH-terminal deletion mutant of p65 (p65ΔC) failed to reduce the expression levels of p73α, suggesting that NF-κB-mediated proteolytic degradation of p73α requires the transcriptional activity of NF-κB. Taken together, our present results imply that NF-κB-mediated degradation of proapoptotic p73 is a novel inhibitory mechanism of p73 that regulates cell survival and death.

Neuroblastoma oligo-capping cDNA project: toward the understanding of the genesis and biology of neuroblastoma

Neuroblastoma (NBL) is a common pediatric cancer originated from the neuronal precursor cells of sympathoadrenal lineage. NBLs show a variety of clinical phenotypes from spontaneous regression to malignant progression with acquirement of resistance to therapy. To understand the molecular mechanism of the genesis, progression, and regression of NBL, we need to identify key molecules determining the neuronal development of sympathoadrenal lineage. To this end, we have performed the NBL cDNA project. It includes (1) mass-cloning of the expressed genes from oligo-capping cDNA libraries derived from primary NBLs with different clinical and biological features; (2) mass-identification of differentially expressed genes between favorable and unfavorable subsets; and (3) molecular and functional analyses of the novel genes, which could be useful prognostic indicators. To date, 10,000 cDNA clones in total, approximately 40% of which contained novel sequences, were randomly picked up and DNA sequenced. We have identified approximately 500 differentially expressed genes between favorable and unfavorable subsets of NBL, among which more than 250 were the genes with unknown function.

Prognostic and therapeutic implications of the MIB-1 labeling index in breast cancer

BackgroundAssessment of tumor proliferative activity is considered to be the most powerful prognostic factor aside from axillary lymph node status. The purpose of this study is to assess the clinical value of measurement of proliferative activity using the MIB-1 labeling index in patients with breast cancer.MethodsSurgical specimens from 36 patients with benign breast disorders and 146 patients with breast cancer were investigated. The MIB-1 labeling index was determined on the specimens stained by immunohistochemical methods as much as possible. Clinical factors associated with the MIB-1 labeling index were reviewed.ResultsThe MIB-1 labeling index for non-proliferative disorders, proliferative disorders, and breast cancer was 3.4±1.9%, 8.9±6.2% and 20±12%, respectively. The MIB-1 labeling index and tumor size, lymph node metastasis status, and clinical stage according to the TNM classification correlated significantly. Survival rate was inversely correlated with the MIB-1 labeling index. No patient with an MIB-1 labeling index of less than 10% had lymph node metastases, and all are alive without recurrence. Patients with an MIB-1 labeling index of over 30% had an extremely poor prognosis.ConclusionsThe MIB-1 labeling index is very useful for predicting both either extremely good or extremely poor prognosis, and axillary lymph node metastasis.

Report of a case with a spontaneous mesenteric hematoma that ruptured into the small intestine

Highlights • This article reports an extremely rare case of spontaneous mesenteric hematoma that ruptured into the jejunum. The etiology of this condition was not revealed by detailed pathological examinations.

Report of a case with gallbladder carcinoma: P53 expression of the peritumor epithelium might predict biliary tract recurrence

Highlights • The over-expression of P53 protein in gallbladder carcinoma is a biomarker correlating with a poor survival. However, the significance of P53 expression in peritumor tissues is unknown. We report a case of gallbladder carcinoma where the operative specimen showed over-expression of P53 on the peritumor epithelium, and early recurrence developed at the biliary tract.• The immunohistochemical staining of the GB wall or surgical stump for a surgical specimen of GBC may be crucial to predict the bile duct recurrence.