Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells

We previously demonstrated that the phytosphingosine-induced apoptosis was accompanied by the concomitant induction of both the caspase-8-mediated and mitochondrial activation-mediated apoptosis pathways. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in the activation of these two distinct cell death pathways induced by phytosphingosine in human cancer cells. Phytosphingosine caused strong induction of caspase-8 activity and caspase-independent Bax translocation to the mitochondria. A rapid decrease of phosphorylated ERK1/2 and a marked increase of p38 MAPK phosphorylation were observed within 10 min after phytosphingosine treatment. Activation of ERK1/2 by pretreatment with phorbol 12-myristate 13-acetate or forced expression of ERK1/2 attenuated phytosphingosine-induced caspase-8 activation. However, Bax translocation and caspase-9 activation was unaffected, indicating that down-regulation of the ERK activity is specifically required for the phytosphingosine-induced caspase-8-dependent cell death pathway. On the other hand, treatment with SB203580, a p38 MAPK-specific inhibitor, or expression of a dominant negative form of p38 MAPK suppressed phytosphingosine-induced translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation but did not affect caspase-8 activation, indicating that activation of p38 MAPK is involved in the mitochondrial activation-mediated cell death pathway. Our results suggest that phytosphingosine can utilize two different MAPK signaling pathways for amplifying the apoptosis cascade, enhancing the understanding of the molecular mechanisms utilized by naturally occurring metabolites to regulate cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both our understanding of cell death events and development of cancer therapeutic agents.     

Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex

The mammalian ATM/PI 3-kinase-related TRRAP protein was previously found to be a component of a multi-protein histone acetyltransferase (HAT) complex containing the HAT TIP60. In this report, we identify a previously uncharacterized protein encoded by the FLJ10914 ORF, which we designate MRGBP, as a new component of the TRRAP/TIP60 HAT complex. In addition, through purification of MRGBP and its associated proteins from HeLa cell nuclear extracts, we identify the thyroid receptor coactivating protein (TRCp120), DMAP1, and the related MRG15 and MRGX proteins as MRGBP-associating proteins, and we present biochemical evidence that they are previously unrecognized components of the TRRAP/TIP60 HAT complex. Taken together, our findings shed new light on the structure and function of the mammalian TRRAP/TIP60 histone acetyltransferase complex.     

Leukotactin-1-induced ERK activation is mediated via Gi/Go protein/PLC/PKC delta/Ras cascades in HOS cells

Recently cloned leukotactin-1 (Lkn-1) that belongs to CC chemokine family has not been characterized. To understand the intracellular events following Lkn-1 binding to CCR1, we investigated the activities of signaling molecules in response to Lkn-1 in human osteogenic sarcoma cells expressing CCR1. Lkn-1-stimulated cells showed elevated phosphorylation of extracellular signal-related kinases (ERK1/2) with a distinct time course. ERK activation was peaked in 30 min and 12 h showing biphasic activation of ERK. Pertussis toxin, an inhibitor of G(i)/G(o) protein, and phospholipase C (PLC) inhibitor blocked Lkn-1-induced activation of ERK. Protein kinase C delta (PKC delta) specific inhibitor rottlerin inhibited ERK activation in Lkn-1-stimulated cells. The activities of PLC and PKC delta were also enhanced by Lkn-1 stimulation. Dominant negative Ras inhibited activation of ERK. Immediate early response genes such as c-fos and c-myc were induced by Lkn-1 stimulation. Lkn-1 affected the cell cycle progression by cyclin D(3) induction. These results suggest that Lkn-1 activates the ERK pathway by transducing the signal through G(i)/G(o) protein, PLC, PKC delta and Ras, and it may play a role for cell proliferation, differentiation, and regulation of gene expression for other cellular processes.     

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.     

One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1

The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis. However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature. Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria. The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L). The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.     

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

Role of Ca2+-activated Cl- channels in the mechanism of apoptosis induced by cyclosporin A in a human hepatoma cell line

The mechanism of apoptosis induced by cyclosporin A (CsA) in a human hepatoma cell line was investigated. CsA induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoma cells. CsA induced Cl- efflux, which was significantly blocked by niflumic acid (NA), a specific inhibitor, and flufenamic acid (FA), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), and 4,4'-diisothiocyanoto-stibene-2,2'-disulfonic acid (DIDS), non-specific inhibitors of Ca2+-activated Cl- channels (CaCCs), not by calyculin A, an inhibitor of K+,Cl- -cotransport. In addition, CsA did not alter intracellular K+ concentration. Moreover, CsA increased intracellular Ca2+ concentration, and treatment with BAPTA/AM, an intracellular Ca2+ chelator, significantly inhibited the CsA-induced Cl- efflux, indicating that CsA induced Cl- efflux through the activation of CaCCs. Treatment with these CaCC inhibitors (NA, FA, NPPB, and DIDS) markedly prevented the CsA-induced apoptosis. Taken together, these results suggest that CaCCs may mediate apoptosis induced by CsA in HepG2 cells. Furthermore, these results provide a new insight into the novel function of CaCCs in the regulation of cancer cell apoptosis associated with perturbation of intracellular Ca2+ signal.     

Effect of culture media on the chemical and physical characteristics of polysaccharides isolated from Poria cocos mycelia

Mycelia of a wild strain Poria cocos were cultured in two media differing in one constituent: bran extract or corn steep liquor, and are designated as wb and wc, respectively. Six polysaccharide fractions were isolated sequentially from the two mycelia by 0.9% NaCl (PCM1), hot water (PCM2), 0.5 M NaOH (PCM3-I and -II) and 88% formic acid (PCM4-I and -II). Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), 13C NMR, light scattering (LS) and viscometry. The results indicated that wb-, wc-PCM1, and PCM2 were heteropolysaccharides mainly composed of alpha-D-glucose, mannose, and galactose, whereas wb-PCM3-I and wc-PCM3-I were mainly (1-->3)-alpha-D-glucans, and wb- and wc-PCM3-II, PCM4-I and PCM4-II were (1-->3)-beta-D-glucans. Interestingly, (1-->3) alpha- and (1-->3)-beta-D-glucans co-existed in the 0.5 M NaOH fraction and were separated individually into the two fractions (PCM3-I and PCM3-II) after neutralizing with acetic acid. The polysaccharides from wc-PCM cultured in media containing corn steep liquor contained relatively more protein. The polysaccharide fractions also existed in conformations including random coil (as in PCM0 and PCM1) and expanded chain (as in PCM3), and differed molecular mass. In addition, two exo-polysaccharides isolated from the two culture media by methanol precipitation (wb- and wc-PCM0) also differed in their monosaccharide composition.     

Cranial neural crest-derived mesenchymal proliferation is regulated by Msx1-mediated p19(INK4d) expression during odontogenesis

Neural crest cells are multipotential progenitors that contribute to various cell and tissue types during embryogenesis. Here, we have investigated the molecular and cellular mechanism by which the fate of neural crest cell is regulated during tooth development. Using a two- component genetic system for indelibly marking the progeny of neural crest cells, we provide in vivo evidence of a deficiency of CNC-derived dental mesenchyme in Msx1 null mutant mouse embryos. The deficiency of the CNC results from an elevated CDK inhibitor p19(INK4d) activity and the disruption of cell proliferation. Interestingly, in the absence of Msx1, the CNC-derived dental mesenchyme misdifferentiates and possesses properties consistent with a neuronal fate, possibly through a default mechanism. Attenuation of p19(INK4d) in Msx1 null mutant mandibular explants restores mitotic activity in the dental mesenchyme, demonstrating the functional significance of Msx1-mediated p19(INK4d) expression in regulating CNC cell proliferation during odontogenesis. Collectively, our results demonstrate that homeobox gene Msx1 regulates the fate of CNC cells by controlling the progression of the cell cycle. Genetic mutation of Msx1 may alternatively instruct the fate of these progenitor cells during craniofacial development.