Ken-ichi Isobe

Both Sp1 and Sp3 are responsible for p21waf1 promoter activity induced by histone deacetylase inhibitor in NIH3T3 cells

Histone deacetylase inhibitor‐induced expression of p21WAF1 is p53 independent. In the present study, we provide evidence that trichostatin A (TSA), a specific inhibitor of histone deacetylase, can elevate H3 and H4 acetylation and p21WAF1 expression in NIH3T3 cells at first. To identify the transcription factor which is responsible for histone deacetylase inhibitor‐induced expression of p21WAF1 and understand the potential events occurred during this process, we analyze the response of the mouse p21WAF1 promoter to TSA in detail. The region responsive to TSA treatment in the p21 promoter is located −100 bp upstream from transcription initiation site and contains a GC‐box. The mutation introduced into this GC‐box decreases most of the basal and TSA‐induced promoter activity. The results from gel‐shift assay show that Sp1 and Sp3 bind to this GC‐rich region. Cotransfection with Sp1 and/or Sp3 expression constructs elevate both basal and induced promoter activity, and this elevation is dependent on the present of the GC‐box. By contrast, cotransfection with reverse oriented Sp1 or Sp3 cDNA decreased basal and induced‐promoter activity, as well as GC‐box dependency. These findings provide physical and functional evidence which strongly indicated that both Sp1 and Sp3 are responsible for TSA‐induced transactivation of the murine p21WAF1 promoter in NIH3T3 cells. J. Cell. Biochem. 73:291–302, 1999.

Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148+/− mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148+/− tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148+/− embryonic stem cells and in fetal germ cells from chimeric Zfp148+/− embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.

New transcutaneous jaundice device with two optical paths.

Abstract Objectives: To confirm the accuracy and precision of transcutaneous bilirubin (TcB) values measured by a new device with two optical paths (JM-103) and the value of total serum bilirubin (TSB) level in clinical units of measurement. Methods: For comparison of the levels of accuracy and precision of JM-103 and the old device (JM-102), serum samples were collected from 77 Japanese infants in three different hospitals including 24 preterm infants and 53 term infants. Measurement of TcB by JM-103 and JM- 102 were performed on the forehead of each infant within 30 min before or after blood sampling. Results: The range of TSB was limited to 19.6 mg/dL and to 17 mg/dL for preterm infants. The correlation coefficients for all subjects (r = 0.94) and for term and preterm subjects between TcB measured by JM-103 and TSB was higher than that between TcB measured by JM-102 and TSB. The regression line in term infants between TcB measured by JM-103 and TcB was similar to that in preterm infants. The error distribution of TcB measured by JM-102 and TSB for all subjects (0.00±2.21) and for term and preterm subjects was larger than that of TcB measured by JM-103 and TSB (all subjects, 0.30±1.55).

Why (−)deprenyl prolongs survivals of experimental animals: Increase of anti-oxidant enzymes in brain and other body tissues as well as mobilization of various humoral factors may lead to systemic anti-aging effects

(--)Deprenyl, a monoamine oxidase B (MAO B) inhibitor is known to upregulate activities of anti-oxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) in brain dopaminergic regions. The drug is also the sole chemical which has been repeatedly shown to increase life spans of several animal species including rats, mice, hamsters and dogs. Further, the drug was recently found to enhance anti-oxidant enzyme activities not only in brain dopaminergic regions but also in extra-brain tissues such as the heart, kidneys, adrenal glands and the spleen. We and others have also observed mobilization of many humoral factors (interferone (INF)-gamma, tumor necrosis factor (TNF)-alpha, interleukine (IL)-1beta,2,6, trophic factors, etc.) and enhancement of natural killer (NK) cell functions by (-)deprenyl administration. An apparent extension of life spans of experimental animals reported in the past may be better explained by these new observations that (-)deprenyl upregulate SOD and CAT activities not only in the brain but also in extra-brain vital organs and involve anti-tumorigenic as well as immunomodulatory effect as well. These combined drug effects may lead to the protection of the homeostatic regulations of the neuro-immuno-endocrine axis of an organism against aging.

A NF-?B p65 subunit is indispensable for activating manganese superoxide: Dismutase gene transcription mediated by tumor necrosis factor-?

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by tumor necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), and lipopolysaccharide (LPS). Recently, a TNF‐responsive element (TNFRE) was identified within the second intron of the murine Mn‐SOD gene. The 5′ CCAAT/enhancer binding protein (C/EBP)‐related region within the TNFRE was responsive to TNF, whereas the 3′ NF‐κB‐related region alone was not. This report describes the minimal promoter region of the Mn‐SOD gene and investigates the cis‐acting elements and trans‐acting factors responsible for TNF‐α‐induced Mn‐SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF‐α stimulation, p50 and p65 NF‐κB subunits bound specifically to the newly identified NF‐κB transcription factor‐binding site, distinct from the previously described NF‐κB site, within the intronic enhancer region. In addition, site‐directed mutagenesis and cotransfection studies demonstrated that the NF‐κB p65 subunit enhanced the transcriptional activity of the Mn‐SOD gene through the newly identified NF‐κB site. These results show that a NF‐κB p65 subunit is mainly involved in the molecular mechanisms controlling TNF‐α‐mediated Mn‐SOD gene transcription. J. Cell. Biochem. 77:474–486, 2000.

Cooperative interaction of NF-κB and C/EBP binding sites is necessary for manganese superoxide dismutase gene transcription mediated by lipopolysaccharide and interferon-γ

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by pro‐inflammatory cytokines. We investigated the cis‐acting elements within a tumor necrosis factor‐responsive element (TNFRE) which was identified in the second intron of the murine Mn‐SOD gene. Site‐directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF‐κB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis‐acting elements may be required for induction of Mn‐SOD gene expression mediated by lipopolysaccharide and interferon‐γ.

GADD34 induces p53 phosphorylation and p21/WAF1 transcription

Recently, others and we have shown that one of the functions of GADD34 is a recovery from a shutoff of protein synthesis induced by endoplasmic reticulum stress. GADD34 has been shown to induce growth arrest and apoptosis. Main protein of apoptosis is p53, especially phosphorylation of p53. And one of the main proteins of growth arrest is p21/WAF1. Here we analyzed the effects of GADD34 on p53 phosphorylation and p21/WAF1 transcription. Transfected Myc‐tagged p53 was dose‐dependently phosphorylated at Ser15 by increasing the amount of GADD34. Transfection of GADD34 also induced the endogenous phosphorylation of p53 and enhanced p21 protein expression. Transfection of GADD34 induced p21/WAF1 promoter activity. This activity was dependent on p53, because GADD34 transfection to p53‐deficient cells produced only a slight increase of p21/WAF1 promoter activity. The p21/WAF1 promoter activity was greatly enhanced by the transfection of p53. Both GADD34 and p53 transfection induced much higher p21/WAF1 promoter activity. The promoter activity of p21/WAF1 was very low in GADD34 deficient MEF. The transfection of GADD34 increased the p21/WAF1 promoter activity in GADD34 deficient MEF.

Early growth responsive-1-dependent manganese superoxide dismutase gene transcription mediated by platelet-derived growth factor

Manganese superoxide dismutase (Mn‐SOD) plays a major role in protecting mitochondria from oxidative damage. Overexpression of Mn‐SOD maintains cell survival under conditions that lead to apoptotic death. In addition to the antioxidative enzyme, platelet‐derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation by activating survival signaling pathways in various cells. Here we show that PDGF induced the expression of the Mn‐SOD gene in NIH3T3 cells, and its induction was associated with early growth response‐1 (Egr‐1), a transcription factor. An electrophoretic mobility shift assay demonstrated that Egr‐1 bound to the proximal promoter of the Mn‐SOD gene in response to PDGF. The proximal promoter region of Mn‐SOD was shown to be transcriptionally responsive to both basal and PDGF stimulation by transfection studies. Forced expression of Egr‐1 in the cells activated Mn‐SOD transcription in a dose‐dependent manner. The pathway by which PDGF induced Egr‐1 involved the mitogen‐activated protein kinase kinase‐1 (MEK1) and extracellular signal‐regulated kinases 1 and 2 (ERK1/2), because the effect of PDGF on the induction of Egr‐1 was blocked by U0126, a specific MEK1 inhibitor. These findings indicate that the induction of Mn‐SOD is part of the anti‐apoptotic properties mediated by PDGF.

Cancer incidence in old age.

The incidence of all cancer increases with age through most of the human life span, but its real incidence at very old ages has not been well elucidated to date. Clarification of the real incidence of cancer in old age, especially among centenarians, may well provide pivotal information to understand the characteristics of humankind. In this study, autopsy records of the Annual of the Pathological Autopsy Cases in Japan, 1991-1996, vols. 34-39 (Japanese Society of Pathology, Tokyo) were used. Cases over 90 years old were studied individually for accurate analysis. The incidence of cancer peaked in the 6th decade and that of multiple cases in the 8th decade. In groups over 90 years of age, the incidence at 5-year intervals did not show any significant decrement. Moreover, the metastatic rate and rate of death due to cancer among centenarians was about three-fourths and two-thirds, respectively, of that of cases aged 90-94 years. The decrease in the metastatic ratio and less mortality due to cancer occurring at the oldest ages are considered due to the nature of cancer itself. The fact that the incidence of cancer does not increase would suggest that certain people among those of advanced age have a special resistance to it.

Cu/Zn- and Mn-superoxide dismutases are specifically up-regulated in neurons after focal brain injury.

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up-regulation of Cu/Zn-SOD only in neurons outside the boundary area, whereas up-regulation of Mn-SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn- and Mn-SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn-SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury.