Hiroshige Hibasami

Dicyclohexylamine, a potent inhibitor of spermidine synthase in mammalian cells

Much effort has been made to develop specific inhibitors of polyamine synthesis which may help in elucidating the role of polyamines in cellular metabolism and in cell proliferation in particular [l-7]. Furthermore, inhibitors of polyamine synthesis may find applications as antiproliferative agents. Four enzymes are involved in the synthesis of polyamines in eukaryotic cells, i.e., ornithine decarboxylase, S-adenosylmethionine(SAM) decarboxylase and two aminopropyltransferases, one catalyzing the synthesis of spermidine, the other producing spermine [8-121. Most inhibitory used are inhibitors of the two decarboxylases, whereas several inhibitors are reported at the aminopropyltransferases [ 13-151. The relative activities of these enzymes must determine which of the polyamines accumulate in the cells. Marked differences in the spermidine to spermine ratio have been observed in comparisons of various mammalian cells [8,16] and spermidine levels are usually elevated in response to growth-promoting stimuli [8,16,17]. Here we demonstrate that partially purified spermidine synthase from rat ventral prostate was strongly inhibited by dicyclohexylamine and also by cyclohexylamine, and that administration of dicyclohexylamine caused a decrease in the concentration of spermidine but not spermine in the liver of partially hepatectomized rats. These inhibitors may be useful as experimental tools for elucidating the physiological function of the polyamines.

Nerve growth factor signaling of p75 induces differentiation and ceramide-mediated apoptosis in Schwann cells cultured from degenerating nerves

In peripheral nerve regeneration or remyelination, immature Schwann cells expressing p75NTR play cardinal roles in the support and regeneration of axons (Griffin JW, Hoffman PN. Peripheral Neuropathy 361–376, 1993). Only one of four to six Schwann cells participate in remyelination of damaged or regenerating axons. The rest of the cells, or supernumerary Schwann cells, show severe atrophy and gradually decrease in number, reestablishing a 1:1 axon–Schwann cell relationship (Said G, Duckett S. Acta Neuropathol (Berl) 53:173–179, 1981). Recent reports demonstrated that severely atrophied supernumerary Schwann cells are eliminated by apoptosis during axonal regeneration or remyelination (Hirata H, Hibasami H. Apoptosis 3:353–360, 1998; Berciano MT, Calle E. Acta Neuropathol (Berl) 95:269–279, 1998). The mechanism to induce selective death of supernumerary Schwann cells without causing any damage to axon‐associated Schwann cells or axons remains to be determined. In this article, we report that p75NTR, the low‐affinity receptor for all members of neurotrophins, signals both cell differentiation and apoptosis through intracellular ceramide elevation. The final response is dependent on the intracellular ceramide level and Schwann cells modulate their response by changing expression level of p75NTR. This effect was selective for nerve growth factor (NGF). Taken together, the present study suggests that NGF contributes both to phenotypic regulation and to elimination of the dedifferentiated Schwann cells, while supporting survival or regeneration of certain types of axons during peripheral nerve repair or regeneration. GLIA 36:245–258, 2001.

Differentiation and apoptosis without DNA fragmentation in cultured Schwann cells derived from wallerian-degenerated nerve

The Schwann cell cables provide particularly favorable sites for the growth of regenerating axonal sprouts. However, if they remain denervated, endoneurial fibrosis takes place with the Schwann cells atrophying and total Schwann cell number gradually decrease with time. Even when regenerating axonal sprouts invade into the cables, Schwann cells do not survive for long periods if they fail to make axonal contact. These observations strongly suggest the involvement of apoptosis in peripheral nerve degeneration and regeneration. So, we investigated the behavior of Schwann cells prepared from walleriandegenerated adult rat sciatic nerve in vitro. The secondary cultured Schwann cells showed serial changes in morphology, mitotic activity and migratory activity as they do during Schwann cell cable formation in vivo. At the final stage of differentiation, the Schwann cells became rounded and detached from the flask with extensive blebbing. Electron micrographs clearly demonstrated typical cytoplasmic changes of apoptosis, but, nuclei of most of the cells retained their size and morphology with residual nucleolar structures. An agarose gel electrophoresis of DNA clearly demonstrated that there was not any DNA fragmentation up to 120 h after detachment. Results by in situ apoptosis detection assay did not show any DNA degradation despite the substantial decrease in Schwann cell number. In conclusion, during peripheral nerve degeneration and regeneration, supernumerary Schwann cells are removed by apoptosis, however, it lacks most of the nuclear events of usual apoptosis.

Effect of zinc administration on pancreatic regeneration after 80% pancreatectomy.

We studied the effect of oral zinc administration on functional and morphological regeneration of the remnant pancreas after 80% pancreatectomy in dogs. After 80% pancreatectomy, both endo- and exocrine function (assessed by the sum of plasma immunoreactive insulin on the intravenous glucose tolerance test and amylase output on the cerulein-secretin test) markedly deteriorated, the pancreatic regeneration rate (change in weight of the remnant pancreas between the time of surgery and autopsy) was very poor, and the zinc concentration in pancreatic tissue decreased in dogs fed the standard diet. In dogs fed the high-zinc diet, pancreatic function and regeneration rate were significantly improved, and the zinc concentration in pancreatic tissue was maintained. Early cell proliferation (assessed by ornithine decarboxylase activity, DNA synthesis, and proliferating cell nuclear antigen labeling index in the remnant pancreas) after pancreatectomy was significantly enhanced in the high-zinc diet group compared to the standard diet group. Correlation analyses between parameters of early cell proliferation and zinc concentration in pancreatic tissue yielded significant positive correlations, and the zinc concentration in pancreatic tissue was significantly correlated with both endo- and exocrine function and the pancreatic regeneration rate. These results suggest that a high-zinc diet after major pancreatectomy is effective in maintaining the zinc concentration in pancreatic tissue, which not only enhances early cell proliferation in the remnant pancreas but improves pancreatic endo- and exocrine function in the late period, promoting pancreatic regeneration.

Purification by affinity chromatography and characterization of porcine liver cytoplasmic polyamine oxidase

1. Polyamine oxidase was purified from the soluble fraction of porcine liver by more than 70,000-fold to electrophoretic homogeneity using N8-acetylspermidine-Sepharose 4B affinity chromatography. 2. The molecular weight and isoelectric point of this enzyme were 62,000 and pH 4.5, respectively. 3. Optimal pH for the catalytic activity was close to 10.0. 4. The enzyme activity was enhanced by 5 mM dithiothreitol or 5 mM benzaldehyde. 5. Preferential substrates for this cytoplasmic PAO were N1-acetylspermine, N1-acetylspermidine and spermine. 6. Spermidine was not virtually the substrate for this enzyme. 7. The present results suggested the physiological roles of cytoplasmic PAO, being coupled with the reaction of spermidine/spermine N1-acetyltransferase, in recycling the cellular polyamines to putrescine.

Induction of spermidine/spermine N1-acetyltransferase in needle-punctured rat lens as a model of traumatic cataract

Isolated rat lens was punctured with a needle at a single point in the equatorial region and was incubated at 37 degrees C. Spermidine/spermine N1-acetyltransferase activity was increased about 5-fold at 8 h after the puncture. Concomitantly, putrescine content in the lens increased markedly at 8-16 h after the puncture, while spermidine levels were slightly depressed. Pretreatment of the lens with actinomycin D or cycloheximide blocked the increases of spermidine/spermine N1-acetyltransferase activity and putrescine content. Ornithine decarboxylase, on the other hand, was not induced to a detectable degree by this stimulus and 5 mM difluoromethylornithine could not block the increase of putrescine content. Polyamine oxidase showed a relatively constant activity that was sufficient for the metabolism of newly formed N1-acetylspermidine. These results suggested that, in the punctured lens, the polyamine levels were regulated predominantly by the activity of spermidine/spermine N1-acetyltransferase, but not by the induction of ornithine decarboxylase.

Antitumor effect of dicyclohexylammonium sulfate, a potent inhibitor of spermidine synthase against P388 leukemia.

The antitumor effect of dicyclohexylammonium sulfate (DCHA), a potent inhibitor of spermidine synthase, was tested on BDF1 mice inoculated i.p. with P388 leukemia (1 X 10(6) cells/mouse). DCHA prolonged the survival time of mice bearing P388 leukemia at the doses of 10-100 mg/kg administered daily for 6 days. The spleen weight increased by 30% at 7 days after tumor inoculation. DCHA treatment had no effect on the tumor-induced increase in splenic weight. The spermidine concentration of the ascites tumor cells and spleens of mice bearing the tumor was lowered by the treatment, while spermine concentration hardly changed. The depletion of spermidine in the ascites tumor cells and spleens might be a cause of the suppression of tumor growth.

Induction of apoptotic cell death in human hepatocellular carcinoma SK-HEP-1 cells by a polyamine synthesis inhibitor, methylglyoxal bis(cyclopentylamidinohydrazone).

The antitumor effects of a polyamine biosynthetic pathway inhibitor methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP) on the human hepatocellular carcinoma SK-HEP-1 cell line have been investigated. The growth of these cultured hepatocellular carcinoma cells was inhibited by MGBCP in a dose-dependent manner. Spermidine and spermine levels were dose-dependently depressed, and morphological changes due to programmed cell death (apoptosis) were observed in these MGBCP-treated hepatocellular carcinoma cells. These results suggest that in addition to reducing the growth rates, MGBCP can induce apoptotic cell death in this human hepatocellular carcinoma cell line.

Chalcones from Angelica keiskei Induce Apoptosis in Stomach Cancer Cells

ABSTRACT Apoptosis was observed in human stomach cancer KATO III cells exposed to two chalcones isolated from the stems of ashitaba (Umbelliferae, Angelica keiskei). Exposure of the KATO III cells to the chalcones, identified by mass spectrometry (MS) and 1H-NMR to be xanthoangelol and 4-hydroxyderricin, produced oligonucreosomal-sized fragments, a characteristic of apoptosis. The DNA fragmentation of the KATO III cells could be observed at concentration of 10 μg L-1 at 2 days after the addition of the chalcones to a culture of KATO III cells, fragmented DNA of human stomach cancer KATO III cells. These findings suggest that growth inhibition by xanthoangelol and 4-hydroxyderricin results from the induction of apoptosis by these chalcones.

Human promyelocytic cell line HL60 has the specific binding sites for prolactin and its ornithine decarboxylase, DNA synthesis and cellular proliferation are induced by prolactin

Human prolactin (hPRL) induced ornithine decarboxylase (ODC) activity, subsequently DNA synthesis and cellular proliferation on human promyelocytic cells, HL60, cultured in a serum-free medium. HL60 cells had 2100 specific binding sites for hPRL per cell, showing a dissociation constant of 1.1 x 10(-10) M. Binding of 125I-PRL to the cells was not blocked by simultaneous addition of human growth hormone. ODC activity and DNA synthesis were activated maximally at 5 and 20 h, respectively, after the addition of 0.05 nM hPRL. These effects of PRL on cellular proliferation, ODC activity and DNA synthesis were abolished by the simultaneous addition of anti-hPRL antibody. Simultaneous addition of an irreversible inhibitor of ODC, difluoromethyl ornithine (DFMO), also abolished the inductions of ODC and DNA synthesis by hPRL. The inhibitory effect of DFMO on hPRL-induced DNA synthesis was reversed by the addition of putrescine to the culture medium. These results suggest that hPRL binds to the prolactin receptor on HL60 cells and induces ODC activity to increase cellular polyamine levels, which eventually stimulates DNA synthesis and cellular proliferation.