Potent inhibition of cell density-dependent apoptosis and enhancement of survival by dimethyl sulfoxide in human myeloblastic HL-60 cells

Human myeloblastic cell line HL-60 cells undergo apoptosis during in vitro culture in a cell density-dependent manner, and this cell density-dependent apoptosis was observed when the concentration of cultured cells exceeded 8–10 × 10⁵ cells/ml. Dimethyl sulfoxide (DMSO), a differentiation inducer of HL-60 cells, did not amplify, but rather potently inhibited, this apoptosis. In a low density culture condition, DMSO attenuated proliferation of HL-60 cells in spite of its inhibition of apoptosis. In contrast, DMSO did support cell survival under high cell density conditions, and DMSO-treated HL-60 cells reached an extremely high concentration of 2–3 × 10⁶ cells/ml, a condition which could never be possible in a usual culture environment. Thus, DMSO exerted dual effects on cell proliferation, i.e., growth inhibition and apoptosis inhibition, and the sum of these effects resulted in an apparently distinct phenomenon according to the culture conditions including cell density. J. Cell. Physiol. 174:135–143, 1998. © 1998 Wiley-Liss, Inc.     

A New Mouse Allele of Glutamate Receptor Delta 2 with Cerebellar Atrophy and Progressive Ataxia

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)–PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.     

Identification of RET autophosphorylation sites by mass spectrometry

The catalytic and signaling activities of RET, a receptor-type tyrosine kinase, are regulated by the autophosphorylation of several tyrosine residues in the cytoplasmic region of RET. Some studies have revealed a few possible autophosphorylation sites of RET by [(32)P]phosphopeptide mapping or by using specific anti-phosphotyrosine antibodies. To ultimately identify these and other autophosphorylation sites of RET, we performed mass spectrometry analysis of an originally prepared RET recombinant protein. Both the autophosphorylation and kinase activity of myelin basic protein as an external substrate of the recombinant RET protein were substantially elevated in the presence of ATP without stimulation by a glial cell line-derived neurotrophic factor, a natural ligand for RET. Mass spectrometric analysis revealed that RET Tyr(806), Tyr(809), Tyr(900), Tyr(905), Tyr(981), Tyr(1062), Tyr(1090), and Tyr(1096) were autophosphorylation sites. Levels of autophosphorylation and kinase activity of RET-MEN2A (multiple endocrine neoplasia 2A), a constitutively active form of RET with substitution of Tyr(900) by phenylalanine (Y900F), were comparable with those of original RET-MEN2A, whereas those of the mutant Y905F were greatly decreased. Interestingly, those of a double mutant, Y900F/Y905F, were completely abolished. Both the kinase activity and transforming activity were impaired in the mutants Y806F and Y809F. These results provide convincing evidence for both previously suggested and new tyrosine autophosphorylation sites of RET as well as for novel functions of Tyr(806), Tyr(809), and Tyr(900) phosphorylation in both catalytic kinase activities and cell growth. The significance of the identified autophosphorylation sites in various protein-tyrosine kinases registered in a data base is discussed in this paper.     

Angiotensin II type 1 receptor interaction is an important regulator for the development of pancreatic fibrosis in mice

The renin-angiotensin system (RAS) plays important roles in various pathophysiological processes. However, the role of the RAS in pancreatic fibrosis has not been established. We investigated the role of angiotensin II (ANG II)-ANG II type 1 (AT(1)) receptor pathway in the development of pancreatic fibrosis with AT(1a) receptor-deficient [AT(1a)(-/-)] mice. To induce pancreatic fibrosis, AT(1a)(-/-) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 microg/kg body wt cerulein at hourly intervals, per week, for four consecutive weeks. Pancreatic fibrosis was assessed by histology and hydroxyproline content. Pancreatic stellate cell (PSC) activation and the localization of AT(1) receptors were assessed by Western blot analysis for alpha-smooth muscle actin and immunostaining. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression in the pancreas was assessed by RT-PCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both AT(1a)(-/-) and WT mice. There were no significant differences between two groups with regard to serum amylase and histological changes. Pancreatic fibrosis induced by repeated episodes of acute pancreatitis was significantly attenuated in AT(1a)(-/-) mice compared with that in WT mice. This finding was accompanied by a reduction of activated PSCs. Dual-immunofluorescence staining in WT mice revealed that activated PSCs express AT(1) receptors. The level of TGF-beta(1) mRNA was lower in AT(1a)(-/-) mice than in WT mice. Our results demonstrate that the ANG II-AT(1) receptor pathway is not essential for the local pancreatic injury in acute pancreatitis but plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation.     

Prostaglandin E2 induced caspase-dependent apoptosis possibly through activation of EP2 receptors in cultured hippocampal neurons

Cyclooxygenase-2 (COX-2) induction and prostaglandin E2 elevation have been reported to occur after cerebral ischemic insult. To evaluate whether the cyclooxygenase-2 reaction product prostaglandin E2 is directly related to induction of apoptosis in neuronal cells, the effect of prostaglandin E2 on cell viability was examined in hippocampal cells. Prostaglandin E2 (5-25 microM) induced apoptosis in a dose-dependent manner 48 h after addition to the cells, which was characterized by cell shrinkage, nuclear condensation or fragmentation and attenuated by a protein synthesis inhibitor, cycloheximide. Neither 17-phenyl trinor-prostaglandin E2 (an EP1 agonist) nor sulprostone (an EP3 agonist) induced cell death, whereas butaprost (an EP2 agonist) induced apoptosis. Prostaglandin E2 increased the intracellular concentration of cAMP, and the selective EP2 agonist butaprost also induced apoptosis accompanied by increasing cAMP levels in hippocampal cells. Moreover, a cell permeable cAMP analog, dibutyryl cAMP also induced apoptosis in hippocampal cells. These findings suggest that prostaglandin E2-induced apoptosis was mediated through a mechanism involving the cAMP-dependent pathway. In addition, prostaglandin E2 activated caspase-3 activity in a dose-dependent manner and a caspase-3 inhibitor prevented the prostaglandin E2-induced apoptosis. We showed in this report that prostaglandin E2 directly induced apoptosis in hippocampal neurons. Moreover, it is likely that the direct effects of prostaglandin E2 on hippocampal neurons were mediated by activation of EP2 receptors followed by elevation of the intracellular cAMP levels.     

Liposomal lactoferrin induced significant increase of the interferon-alpha (IFN-alpha) producibility in healthy volunteers

Interferon-alpha (IFN-alpha) producibility has been widely accepted as one of the important markers to evaluate the immune status. In this study, preliminary clinical tests were carried out to confirm the immunomodulatory activity of liposomal lactoferrin including IFN-alpha producibility and NK activity. In a primary open trial, the liposomal lactoferrin was administered to five healthy males for one week and various immunological indices were evaluated. Furthermore, ten healthy males were administered 319 mg per day of liposomal or non-liposomal lactoferrin for four weeks, and immune status was monitored at 0, 1 and 4 weeks after the intake as well as three weeks after stopping it. In this double-blinded comparative study, the IFN-alpha producibility was significantly increased only in the liposomal lactoferrin group during administration and decreased 3 weeks after stopping it, while the IFN-alpha producibility was unchanged in the non-liposomal lactoferrin group. Although the biological mechanism of IFN-alpha producibility enforced by liposomal lactoferrin has not been wholly understood, it is suggested to be a novel active constituent having preventive and therapeutic effects on inflammatory diseases, cancer and infectious diseases such as chronic hepatitis C.