p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate

BACKGROUND: p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53-responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti-cancer therapy. METHODS: In this study, we adenovirally transduced p51A cDNA into human lung, gastric and pancreatic cancer cells and analyzed the intracellular function of p51 in anti-oncogenesis in vitro and in vivo. RESULTS: Overexpression of p51A revealed an anti-proliferative effect in vitro in all the cancer cells examined in this study. The anchorage-dependent and -independent cell growth of EBC1 cells carrying mutations in both p51 and p53 was suppressed and significant apoptosis following adenoviral transduction with p51 and/or p53 was seen. This growth suppression was cooperatively enhanced by the combined infection with adenoviral vectors encoding both p51 and p53. Furthermore, p51 activated several, but not all, p53-inducible genes, indicating that the mechanisms controlling p51- and p53-mediated tumor suppression differed. CONCLUSIONS: Our observations indicate that, although p51 exhibited reduced anti-oncogenetic effects compared with p53, it cooperatively enhanced the anti-tumor effects of p53. Our results suggest that p51 functions as a tumor suppressor in human cancer cells in vitro and in vivo and may be useful as a potential tool for cancer gene therapy.     

Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1

Actin-related proteins (Arps) were recently shown to contribute to the organization and regulation of chromatin structures. The nuclear functions of Arps have been investigated principally in budding yeast in which six of the ten Arp subfamilies are localized in the nucleus. In vertebrates, only two isoforms of Arp4 have so far been identified as showing localization to the nucleus. Here we show the predominant nuclear localization of another Arp subfamily, Arp6, in vertebrate cells. Vertebrate Arp6 directly interacted with heterochromatin protein 1 (HP1) orthologs and the two proteins colocalized in pericentric heterochromatin. Yeast Arp6 is involved in telomere silencing, while Drosophila Arp6 is localized in the pericentric heterochromatin. Our data strongly suggest that Arp6 has an evolutionarily conserved role in heterochromatin formation and also provide new insights into the molecular organization of heterochromatin.     

Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.     

Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.     

Construction and expression of anti-Tn-antigen-specific single-chain antibody genes from hybridoma producing MLS128 monoclonal antibody

Anti-Tn-antigen monoclonal antibody MLS128 has affinity for three consecutive Tn-antigens (Tn3) more than Tn2. The major aim of this study was to isolate genes encoding MLS128 variable domains to produce a large quantity of recombinant MLS128 antibodies, in turn, allowing the conduct of studies on precise interactions between Tn3- or Tn2-epitopes and MLS128. This study describes cloning of the variable region genes of MLS128, construction of the variable region genes in single-chain variable fragments (scFv) and two scFvs conjugated with human IgG(1) hinge and Fc regions (scFv-Fc) types, and their respective expression in bacterial and mammalian cell. MLS128 scFv protein with the expected specificity and affinity was successfully prepared from inclusion bodies accumulating in Escherichia coli. Construction, expression and purification of two types of MLS128-scFv-Fc proteins with differing linker lengths in Chinese hamster ovary cells demonstrated that the purified scFv-Fc proteins had binding activity specific to the glycoprotein-expressing Tn-antigen clusters. These results revealed that VL and VH genes cloned from the hybridoma represent those of MLS128 and that recombinant antibodies produced from these genes should provide sufficient amounts of binding domains for use in 3D structural studies such as NMR and X-ray analysis.     

Involvement of AMPA Receptor GluR2 and GluR3 Trafficking in Trigeminal Spinal Subnucleus Caudalis and C1/C2 Neurons in Acute-Facial Inflammatory Pain

To evaluate the involvement of trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR2 and GluR3 subunits in an acute inflammatory orofacial pain, we analyzed nocifensive behavior, phosphorylated extracellular signal-regulated kinase (pERK) and Fos expression in Vi/Vc, Vc and C1/C2 in GluR2 delta7 knock-in (KI), GluR3 delta7 KI mice and wild-type mice. We also studied Vc neuronal activity to address the hypothesis that trafficking of GluR2 and GluR3 subunits plays an important role in Vi/Vc, Vc and C1/C2 neuronal activity associated with orofacial inflammation in these mice. Late nocifensive behavior was significantly depressed in GluR2 delta7 KI and GluR3 delta7 KI mice. In addition, the number of pERK-immunoreactive (IR) cells was significantly decreased bilaterally in the Vi/Vc, Vc and C1/C2 in GluR2 delta7 KI and GluR3 delta7 KI mice compared to wild-type mice at 40 min after formalin injection, and was also significantly smaller in GluR3 delta7 KI compared to GluR2 delta7 KI mice. The number of Fos protein-IR cells in the ipsilateral Vi/Vc, Vc and C1/C2 was also significantly smaller in GluR2 delta7 KI and GluR3 delta7 KI mice compared to wild-type mice 40 min after formalin injection. Nociceptive neurons functionally identified as wide dynamic range neurons in the Vc, where pERK- and Fos protein-IR cell expression was prominent, showed significantly lower spontaneous activity in GluR2 delta7 KI and GluR3 delta7 KI mice than wild-type mice following formalin injection. These findings suggest that GluR2 and GluR3 trafficking is involved in the enhancement of Vi/Vc, Vc and C1/C2 nociceptive neuronal excitabilities at 16-60 min following formalin injection, resulting in orofacial inflammatory pain.     

A strategy for screening an inhibitor of viral silencing suppressors, which attenuates symptom development of plant viruses

To find out whether we can control plant virus diseases by blocking viral RNA silencing suppressors (RSSs), we developed a strategy to screen inhibitors that block the association of RSSs with siRNAs using a surface plasmon resonance assay. The screened chemicals were tested in competition with RSSs for binding to siRNAs using a mobility shift assay. We then confirmed that tested chemicals actually inhibited the RSS activity in vivo using a protoplast assay which was developed for this purpose. This entire system can be adapted to screening inhibitors of not only plant viruses but also some animal viruses possessing RSSs.