Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addition to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 physically binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunoprecipitation analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the physical interaction with Plk1. The ectopically overexpressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21 WAF1 , MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter analysis, Plk1 had an ability to block the p53-dependent induction of the endogenous p21 WAF1 . In addition, Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a critical target of Plk1, and its function is abrogated through the physical interaction with Plk1.

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 NH 2 -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. The BRCA1 carboxyl terminus (BRCT)3 domain is defined by distinct hydrophobic clusters of amino acids and is often found in a variety of cellular proteins such as BRCA1, 53BP1, and RAD9, which are involved in DNA repair and/or DNA damage signaling pathways (1-3). Although the functional role of the BRCT domain is currently unclear, the early works indicated that the BRCT domains of BRCA1 act as a transactivator (4, 5). Consistent with this notion, the point mutations detected within the BRCT domains of BRCA1 markedly inhibited its transcriptional activity (4, 5), and BRCA1 was a component of RNA polymerase II holoenzyme (6). Alternatively, the BRCT domains function as protein-protein interaction modules (7). For example, the BRCT domains of BRCA1 as well as 53BP1 were required for the interaction with p53 (8, 9). NFBD1/MDC1 (nuclear factor with BRCT domain 1/mediator of DNA damage checkpoint protein 1) is a large nuclear protein bearing three characteristic structural domains, including an NH 2 -terminal forkhead-associated (FHA) domain, an internal PST (proline/serine/threonine-rich) repeat domain, and tandem repeat of COOH-terminal BRCT domains (10 -14). We have initially reported that NFBD1 acts as a nuclear transcriptional factor with an anti-apoptotic function (11). In accordance with our results, siRNA-mediated knockdown of NFBD1 led to a significant increase in the number of apoptotic cells (15). In addition, the elimination of NFBD1 expression increased the sensitivity to irradiation (16). These observations indicate that NFBD1 has an anti-apoptotic function; however, the precise molecular mechanisms behind the anti-apoptotic effect of NFBD1 remain to be explored.Previous studies strongly suggest that NFBD1 is closely involved in early cellular responses to genotoxic stress. Upon DNA damage, NFBD1 was phosp...

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 p73alpha containing SAM domain. During the cisplatin-induced apoptosis in SH-SY5Y neuroblastoma cells, p73alpha 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 p73alpha in association with a significant inhibition of the p73alpha-mediated transactivation as well as proapoptotic activity. Downregulation of endogenous UFD2a by antisense strategy resulted in a remarkable accumulation of p73alpha. Unexpectedly, UFD2a-mediated degradation of p73alpha was sensitive to the proteasomal inhibitor, however, UFD2a did not affect the ubiquitination levels of p73alpha. 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.

The extremely high rate (68.8%) of peritoneal metastases was observed in long-term follow-up for the metastatic breast cancer patients with ILC. We need to reveal the definitive feature of ILC and develop new therapeutic strategies to prevent the dissemination of ILCs.

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

In response to DNA damage, p53 and its homolog p73 have a function antagonistic to NF-B in deciding cell fate. Here, we show for the first time that p73, but not p53, is stabilized by physical interaction with nuclear IB kinase (IKK)-␣ to enhance cisplatin (CDDP)-induced apoptosis. CDDP caused a significant increase in the amounts of nuclear IKK-␣ and p73␣ in human osteosarcoma-derived U2OS cells. Ectopic expression of IKK-␣ 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 RNAmediated knockdown of endogenous IKK-␣ inhibited the CDDP-mediated accumulation of p73␣. The kinase-deficient mutant form of IKK-␣ interacted with p73␣, but failed to stabilize it. Furthermore, CDDP-mediated accumulation of endogenous p73␣ was not detected in mouse embryonic fibroblasts (MEFs) prepared from IKK-␣-deficient mice, and CDDP sensitivity was significantly decreased in IKK-␣-deficient MEFs compared with wild-type MEFs. Thus, our results strongly suggest that the nuclear IKK-␣-mediated accumulation of p73␣ 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.

Breast-conserving oncoplastic surgery carries a risk of fat necrosis as a delayed complication. The incidence rate and severity of fat necrosis with each procedure should be assessed. We should select fat grafts with a good blood supply to replace defects of breast-conserving therapy.

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 ␤ (PKA-C␤) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73␣ associated with PKA-C␤ in mammalian cells and that their interaction was mediated by both the N-and C-terminal regions of p73␣. In contrast, p53 failed to bind to PKA-C␤. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73␣-(1-130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-C␤ resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73␣, whereas a kinase-deficient mutant of PKA-C␤ 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 p73␣. Of particular interest, PKA-C␤ facilitated the intramolecular interaction of p73␣, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-C␤-mediated inhibitory mechanism of p73 function. p73 has been identified as a structural and functional homolog of the tumor suppressor p53 (1). p53 and p73 share the same domain organization, consisting of an N-terminal transactivation domain, a central sequence-specific DNA-binding domain, and a C-terminal oligomerization domain. As expected, several pieces of evidence suggest that p73 can bind to the p53-responsive element and transactivate an overlapping set of p53 target genes, thus leading to induction of G 1 /S cell cycle arrest and apoptosis (1-6). In marked contrast to p53, p73 is expressed as multiple isoforms arising from alternative splicing of the primary p73 transcript (p73␣, p73␤, p73␥, p73␦, p73⑀, p73, and p73) termed the TA variant (1, 3, 7-9). These alternatively spliced isoforms vary in their C termini and display different transcriptional and biological properties. Additionally, the ⌬N variant (⌬Np73␣ and ⌬Np73␤), which is generated by alternative promoter utilization, lacks the N-terminal transactivation domain and exhibits dominantnegative behavior toward wild-type p73 as well as p53 (10 -12). Recently, we (14) and others (13, 15) demonstrated that p73 directly transactivates the expression of its own negative regulator (⌬Np73), creating an autoregulatory feedback loop in which both the activity of p73 and the expression of ⌬Np73 are regulated. Thus, the pro-apoptotic activity of p73 is determined by the relative expression levels of its TAp73 and dominantnegative ⌬Np73 variants in cells.In sharp contrast to p53, it was initially reported that p73 was not induced by DNA damage (1). However, recent studies demonstrated that, in response to a subset of DNA-damaging agents, p73 is positively regu...