Sadao Gotoh

Analysis of N-myc amplification in relation to disease stage and histologic types in human neuroblastomas

Both untreated and treated primary neuroblastomas from 52 patients were analyzed to determine the correlation between the amplification of N‐myc oncogene and various prognostic factors. Amplification of N‐myc was observed in eight of 28 untreated cases and in 12 of 24 treated cases. As a whole, 12 of 18 tumors (67%) in Stage IV had N‐myc amplification, but there were fewer cases in the unadvanced disease stage, as reported previously by others. Furthermore, the authors detected N‐myc amplification in three of nine tumors in Stage IV‐S, although the amplification was less than 50 copies. Analysis of progression‐free survival at 24 months revealed that amplification of N‐myc was associated with the worst prognosis (P < 0.001). In the untreated group, no amplification of N‐myc was detected in any of two ganglioneuromas and four ganglioneuroblastomas, whereas amplification of N‐myc was observed in all two round‐cell and six of 20 rosette fibrillary neuroblastomas. On the other hand, the authors detected amplification of N‐myc in three of eight less differentiated ganglioneuroblastomas in the treated group and observed the worst prognosis in these three patients. The total percentage of the cases from both untreated and treated groups suggest that amplification of N‐myc may occur more frequently in undifferentiated types of neuroblastomas than in less malignant types. In conclusion, the amplification of N‐myc in neuroblastomas was closely associated with the worst prognosis, which was suggested by both disease stage and histologic characteristics.

Adrenomedullin regulates blood-brain barrier functions in vitro.

Adrenomedullin (AM) is an important vasodilator in cerebral circulation, and cerebral endothelial cells are a major source of AM. This in vitro study aimed to determine the AM-induced changes in blood–brain barrier (BBB) functions. AM administration increased, whereas AM antisense oligonucleotide treatment decreased transendothelial electrical resistance. AM incubation decreased BBB permeability for sodium fluorescein (mol. wt 376 Da) but not for Evans blue albumin (mol. wt 67 kDa), and it also attenuated fluid-phase endocytosis. AM treatment resulted in functional activation of P-glycoprotein efflux pump in vitro. Our results indicate that AM as an autocrine mediator plays an important role in the regulation of BBB properties of the cerebral endothelial cells.

Cerebral endothelial cells are a major source of adrenomedullin.

Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase‐polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 ± 13 fmol/105 cells per 12 h; mean ± SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte‐derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose‐dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood–brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.

A garlic lectin exerted an antitumor activity and induced apoptosis in human tumor cells

Cytotoxic effects of a lectin prepared from garlic (Allium sativum-L) bulbs on human tumor cells were studied. The lectin strongly reduced the growth and DNA synthesis of human tumor cells in a time- and a dose-dependent manner. By contrast, a soybean lectin showed only a weak inhibitory effect on growth and DNA synthesis of tumor cells. Furthermore, the garlic lectin induced apoptosis in the cells at a low concentration. The antitumor activity of garlic lectin may provide a rational basis for its effectiveness observed in clinical applications.

Simultaneous activation of heat shock protein (hsp 70) and nucleolin genes during in vivo and in vitro prereplicative stages of rat hepatocytes

Rapidly growing cells usually have high levels of ribosome biogenesis. The sequential expression of protooncogenes during the transition of quiescent hepatocytes to the replicative stage was assumed to be followed by activation of cellular genes related to cell growth such as ribosome biosynthesis. First, the expression of major nucleolar protein (nucleolin or C23) and major heat-shock protein (hsp 70) genes was examined during rat liver regeneration. hsp 70 may function in cell growth and has a characteristic nucleolar location after heat shock. Both nucleolin and hsp 70 mRNA began to increase simultaneously after peaks of c-fos and c-myc, showed a peak 6 h after partial hepatectomy, and declined to the control levels around 20 h. That is, the peaks of nucleolin and hsp 70 mRNA precede the peak of ribosome formation (12-20 h) and DNA replication (24 h). Second, the behavior of nucleolin and hsp 70 mRNA was examined in primary cultured hepatocytes during their G0-G1 transition. Although the amounts of c-myc mRNA reached a plateau around 20 h after the initiation of culture and remained at these levels, DNA synthesis has never been found to start without the addition of EGF and insulin to this system. Both nucleolin and hsp 70 mRNA began to increase at around 20 h (prereplicative stage) and simultaneously decreased in inverse proportion to DNA synthesis induced by these growth factors. Thus, it is possible that the simultaneous enhancement of nucleolin and hsp 70 genes as described above is not merely coincidental, but is important biologically during the transition of quiescent hepatocytes to proliferative cells.

Different responses other than the formation of DNA-adducts between the livers of carcinogen-resistant rats (DRH) and carcinogen-sensitive rats (Donryu) to 3'-methyl-4-dimethylaminoazobenzene administration

Carcinogen‐resistant inbred DRH rats developed from the Donryu strain showed a remarkably low incidence of liver tumors when they were fed diets containing hepatocarcinogens such as 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐Me‐DAB). In this work, we examined various characteristics of male DRH and Donryu rats during 3′‐Me‐DAB administration for 8 weeks. 32P‐Postlabeling analysis showed that essentially similar levels of DNA‐adducts were generated by the metabolites of 3′‐Me‐DAB in the livers of these two strains of rats at several time points. However, both GADD45 (growth arrest and DNA damage‐inducible) and O6‐methylguanine methyltransferase (putatively DNA damage‐inducible) mRNA levels were increased significantly in Donryu rat livers, but were increased to a lesser extent in DRH rats. [3H]Thymidine incorporation into hepatic DNA began to increase around 10 to 20 days after the start of 3′‐Me‐DAB administration in Donryu rats probably due to DNA repair, while no significant change occurred in DRH rats under the same conditions. Furthermore, inductions of heme oxygenase (due to degradation of heme‐proteins) and hepatocyte growth factor (HGF; cell death and regeneration of hepatocytes) mRNAs were greater in Donryu rat livers than those of DRH, suggesting that the former were more sensitive to cytotoxic effects of 3′‐Me‐DAB than the latter. Another remarkable difference observed between these two strains was the significant induction of cytochrome P‐450 2E1 mRNA in Donryu rat livers; this may contribute to the generation of reactive oxygen intermediates. Finally, increases of glutathione S‐transferase (P‐form) and γ‐glutamyltranspeptidase mRNAs as marker enzymes of preneoplastic changes of hepatocytes were clearly seen only in Donryu rat livers at 6 to 8 weeks after the start of 3′‐Me‐DAB administration. These results indicate that the different susceptibility to hepatocarcinogenesis between these two strains of rats may arise from events other than the DNA adduct formation.

Cloning of cDNAs with possible association with senescence and immortalization of human cells

Normal human diploid fibroblasts (HDF) have a finite life span in vitro and have been used as a model system for the study of in vivo aging. Little is known about how changes in gene expression may affect the immortalization of human fibroblasts. We looked for cDNA clones whose mRNAs were differentially expressed between mortal senescent SV40-transformed human fibroblasts (B-32) and the immortal counterparts (B-32F) derived from B-32 cells. We identified three cDNA isolates by subtractive differential hybridization with 32P-labeled cDNA probes from B-32 cells and B-32F cells. Nucleotide sequence analysis of these cDNA clones revealed that they were homologous to the human vimentin, a human mitochondrial gene and a human gene of unknown nature. Slot blot and Northern blot analyses demonstrated that the former two were preferentially expressed in senescent B-32 cells and the last one was less expressed in B-32F immortal cells.

Different gene expression for ribosomal RNA of AH-130 tumor and embryonic and adult rat liver.

Abstract The nucleotides of 45-S ribosomal precursor RNA in the nucleoli of a number of tumors comprise less adenylic, and more cytidylic acid than those of normal rat liver. The base ratios of cytoplasmic mature ribosomal RNA in tumors are rather similar to those in normal rat liver, but the 32 P distribution in oligonucleotides of ribosomal RNA of these tissues differs appreciably. The isotope content of dinucleotides of RNA of tumors was higher, and that of pentanucleotides was lower than in RNA of normal liver. Analyses of digestion products of ribosomal RNA of these tissues suggested that the primary structure of ribosomal RNA of tumors might differ from that of rat liver. Furthermore, the 32 P distribution in the isoplithic peaks of ribosomal RNA of tumor cells were similar to that of 14-day-old embryonic liver. These results suggested that gene expression for ribosomal RNA may differ in adult liver and less differentiated tissues.

Down-regulation of telomerase activity by anticancer drugs in human ovarian cancer cells

Maintenance of telomere length is crucial for survival of cells. Telo-merase, an enzyme that is responsible for elongation of shortened telomeres, is active in human germ cells as well as most tumor tissues and experimentally immortalized cells. In contrast, most mature somatic cells in human tissues express undetectable or low telomerase activity, implying the existence of a stringent and negative regulatory mechanism. In this study we report the effects of anticancer drugs on telomerase activity in human cancer cells. In assaying for telomerase activity, we basically followed the original TRAP assay system, but with some modifications. A down-regulation of telomerase activity was found when cells of a human ovarian cancer cell line, A2780, were treated with;cis-diamminedichloroplatinum(II) (CDDP; cisplatin). However, down-regulation of telomerase activity was not found in cells of a cisplatin-resistant cell line, A2780CP, treated with cisplatin. On the other hand, telomerase activity in both the cell lines A2780 and A2780CP was reduced when A2780 or A2780CP was treated with adriamycin, an anthracycline antibiotic having a broad spectrum of antineoplastic activity. The different effects on the telomerase activity of the two types of anticancer drugs may be due the distinct chemical functions of these drugs. The present results may indicate a positive relationship between anticancer effects and down-regulation of telomerase activity by anticancer drugs.

Genetic analysis of the cell binding domain region of the chicken fibronectin gene

We have determined the nucleotide sequence of the cell binding domain region of the chicken fibronectin gene and analyzed it evolutionaly. We present here the complete nucleotide sequence of 4.3 kb HindIII/EcoRI segment from the clone lambda FC23 of the chicken fibronectin gene. There were five exons in this segment. When we lined up the amino acid of exons 28, 29 and 31, three alignments, known as the Type III repeat, appeared. Tetrapeptide, -RGDS-, called the cell binding domain, existed in the second repeat, coding exon 30. It was presumed that the Type III repeats were composed of two exons in the chicken gene, the same as in the rat and humans. We found repeatedly appearing amino-acid sequences such as -TIT- (three arrays in these Type III repeats) but also found one of the amino acids substituted in the tripeptide in these Type III repeats (seven arrays). We analyzed these repeats from the point of view of evolution. We used three of the nucleotide sequences (12-18 bp) coding such -TIT- repeats as a unit length for comparing the various homologies after dividing the coding region into 56 segments. The mutual homology of the divided segments to each one of three showed 53% on average. On the other hand, the mutual nucleotide homology of the Type III repeat was 44%. This suggested that the Type III repeat may have been developed by frequent duplication of small gene units.