Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells

Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

Sleep-related factors associated with industrial accidents among factory workers and sleep hygiene education intervention

Sleep and Biological Rhythms - This study was conducted to investigate the association between industrial accidents and sleep-related parameters in factory workers, and to examine the effectiveness...     

Background Factors of Reflux Esophagitis and Non-Erosive Reflux Disease: A Cross-Sectional Study of 10,837 Subjects in Japan

Background

Despite the high prevalence of gastroesophageal reflux disease (GERD), its risk factors are still a subject of controversy. This is probably due to inadequate distinction between reflux esophagitis (RE) and non-erosive reflux disease (NERD), and is also due to inadequate evaluation of adjacent stomach. Our aim is therefore to define background factors of RE and NERD independently, based on the evaluation of Helicobacter pylori infection and gastric atrophy.

Methods

We analyzed 10,837 healthy Japanese subjects (6,332 men and 4,505 women, aged 20–87 years) who underwent upper gastrointestinal endoscopy. RE was diagnosed as the presence of mucosal break, and NERD was diagnosed as the presence of heartburn and/or acid regurgitation in RE-free subjects. Using GERD-free subjects as control, background factors for RE and NERD were separately analyzed using logistic regression to evaluate standardized coefficients (SC), odds ratio (OR), and p-value.

Results

Of the 10,837 study subjects, we diagnosed 733 (6.8%) as RE and 1,722 (15.9%) as NERD. For RE, male gender (SC = 0.557, OR = 1.75), HP non-infection (SC = 0.552, OR = 1.74), higher pepsinogen I/II ratio (SC = 0.496, OR = 1.64), higher BMI (SC = 0.464, OR = 1.60), alcohol drinking (SC = 0.161, OR = 1.17), older age (SC = 0.148, OR = 1.16), and smoking (SC = 0.129, OR = 1.14) are positively correlated factors. For NERD, HP infection (SC = 0.106, OR = 1.11), female gender (SC = 0.099, OR = 1.10), younger age (SC = 0.099, OR = 1.10), higher pepsinogen I/II ratio (SC = 0.099, OR = 1.10), smoking (SC = 0.080, OR = 1.08), higher BMI (SC = 0.078, OR = 1.08), and alcohol drinking (SC = 0.076, OR = 1.08) are positively correlated factors. Prevalence of RE in subjects with chronic HP infection and successful HP eradication denotes significant difference (2.3% and 8.8%; p<0.0001), whereas that of NERD shows no difference (18.2% and 20.8%; p = 0.064).

Conclusions

Significantly associated factors of NERD are considerably different from those of RE, indicating that these two disorders are pathophysiologically distinct. Eradication of Helicobacter pylori may have disadvantageous effects on RE but not on NERD.     

DNA polymerase III holoenzyme of Escherichia coli. I. Purification and distinctive functions of subunits tau and gamma, the dnaZX gene products

Escherichia coli dnaZX, the gene which when mutant blocks DNA chain elongation, was cloned into a lambda PL promoter-mediated expression vector. In cells carrying this plasmid, the activity that complements a mutant dnaZ extract in replicating a primed single-stranded DNA circle was increased about 20-fold. Two polypeptides of 71 and 52 kDa were overproduced. Upon fractionation, two complementing activities were purified to homogeneity and proved to be the 71- and 52-kDa polypeptides. Immunoassays revealed their respective identities with the tau and gamma subunits of DNA polymerase III holoenzyme. The N-terminal amino acid sequences of the first 12 residues were identical in both subunits, as were their molar specific activities in dnaZ complementation. Thus, the tau subunit complements the defect in the mutant holoenzyme from the dnaZts strain as efficiently as does the gamma subunit. Inasmuch as the 71-kDa subunit (tau) can also overcome the enzymatic defect in a dnaX mutant strain, this polypeptide has dual replication functions, only one of which can be performed by the gamma subunit. Availability of pure tau and gamma subunits for study has provided the basis for proposing an asymmetry in the structure and function of a dimeric DNA polymerase III holoenzyme.     

Antioxidative Activity of 5,6,7,8-Tetrahydrobiopterin and its Inhibitory Effect on Paraquat-Induced Cell Toxicity in Cultured Rat Hepatocytes

The in vitro antioxidative activity of 5,6,7,8-tetrahydrobiopterin (BPH4) was measured and the ability of BPH4 to prevent paraquat-induced cell damage was examined in cultured hepatocytes. The scavenging activity of BPH4 against superoxide anion radicals was assayed in two systems, i.e., xanthine/xanthine oxidase (X/XOD) and rat macrophage/phorbol myristate acetate (MξPMA) radical-generating systems. BPH4 showed an extremely strong superoxide anion radical-scavenging activity in both assay systems. Biopterin (BP) itself did not show any activity in the X/XOD system, but was effective in the MξPMA system. The antioxidative activities of BPH4 against both superoxide anion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. BPH4 also protected rat brain homogenate against auto-oxidation. We further examined the effect of BPH4 on paraquat-induced cell toxicity in cultured rat hepatocytes. The paraquat-induced elevation of the release of lactate dehydrogenase (LDH), a marker enzyme for cytotoxicity from cultured hepatocytes was suppressed by BPH4 in a dose-dependent manner. The elevation of lipid peroxides simultaneously induced by paraquat was also inhibited by BPH4 in the same manner. These results suggest that BPH4 might be useful in the treatment of various diseases whose pathogenesis is active oxygen-related.     

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.