Positional cloning of the sex-linked giant egg (Ge) locus in the silkworm, Bombyx mori.

The giant egg (Ge) locus is a Z-linked mutation that leads to the production of large eggs. Cytological observations suggest that an unusual translocation of a large fragment of the W chromosome bearing a putative egg size-determining gene, Esd, gave rise to giant egg mutants. However, there is currently no molecular evidence confirming either a W-Z translocation or the presence of Esd on the W chromosome. To elucidate the origin of giant egg mutants, we performed positional cloning. We observed that the Bombyx mori. orthologue of the human Phytanoyl-CoA dioxygenase domain containing 1 gene (PHYHD1) is disrupted in giant egg mutants. PHYHD1 is highly conserved in eukaryotes and is predicted to be a Fe(II) and 2-oxoglutarate-dependent oxygenase. Exon skipping in one of the two available Ge mutants is probably caused by the insertion of a non-long terminal repeat transposon into intron 4 in the vicinity of the 5' splice site. Segmental duplication in Ge(2) , an independent allele, was caused by unequal recombination between short interspersed elements inserted into introns 3 and 5. Our results indicate that (1) Bombyx PHYHD1 is responsible for the Ge mutants and that (2) the Ge locus is unrelated to the W-linked putative Esd. To our knowledge, this is the first report describing the phenotypic defects caused by mutations in PHYHD1 orthologues.

Mitotic catastrophe and cell death induced by depletion of centrosomal proteins.

Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB.

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between -53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

A homolog of the human Hermansky-Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori.

Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa ( 2 ), and oa ( v )), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.

Characterization of an omega-class glutathione S-transferase in the stress response of the silkmoth.

The glutathione S-transferase (GST) superfamily is involved in detoxification of various xenobiotics. Using real-time PCR, mRNA encoding an omega-class GST of Bombyx mori (bmGSTO) was shown to be induced after exposure to various environmental stresses. A soluble form of recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells and purified to homogeneity. Cys 38 and Pro 39 were found to be highly conserved in omega-class GSTs, and their roles were investigated by site-directed mutagenesis/kinetic analysis. Mutations of Cys 38 and Pro 39 residues affected the catalytic efficiency of enzymes, indicating that the presence of Cys 38 and Pro 39 residues is important for bmGSTO activity. Thus, bmGSTO could contribute to increasing the environmental stress resistance of lepidopteran insects.

Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm, Bombyx mori.

The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od(B) ) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ-line transformation of the wild-type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky-Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.

v-Src oncogene product increases sphingosine kinase 1 expression through mRNA stabilization: alteration of AU-rich element-binding proteins.

Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5'-promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.

Implications of sphingosine kinase 1 expression level for the cellular sphingolipid rheostat: relevance as a marker for daunorubicin sensitivity of leukemia cells.

We recently reported increased sphingosine kinase 1 (SPHK1) and decreased neutral sphingomyelinase 2 (NSMase2) gene expression in myelodysplastic syndromes and acute leukemia. This alteration is supposed to change the cellular sphingolipid metabolites; however, positive correlations were observed between daunorubicin (DA)-IC50 and the SPHK1 message but not between DA-IC50 and NSMase2 messages, when 16 different leukemia cell lines were used to analyze the relationship between gene expressions and chemosensitivity against DA. Using two cell lines with either the highest or lowest SPHK1 expression, cellular ceramides and sphingosine 1-phosphate (S1P) were quantified by liquid chromatography/mass spectrometry. Increased ceramide was observed in DA-sensitive, but not in DA-resistant cell lines treated with low doses of DA. Upon DA treatment, S1P decreased more in the sensitive cell lines than in resistant cell lines. A SPHK inhibitor recovered the DA sensitivity of DA-resistant cells. The modulation of SPHK1 gene expression by either overexpression or using siRNA affected the DA sensitivity of representative cell lines. Results clearly show that SPHK1 is both a good marker to predict the DA sensitivity of leukemia cells and a potential therapeutic target for leukemia with high SPHK1 expression, and suggest that the sphingolipid rheostat plays a significant role in DA-induced cytotoxicity.

Identification of the female-determining region of the W chromosome in Bombyx mori.

The W chromosome of the silkworm Bombyx mori is devoid of functional genes, except for the putative female-determining gene (Fem). To localize Fem, we investigated the presence of W-specific DNA markers on strains in which an autosomal fragment containing dominant marker genes was attached to the W chromosome. We produced new W-chromosomal fragments from the existing Zebra-W strain (T(W;3)Ze chromosome) by X-irradiation, and then carried out deletion mapping of these and sex-limited yellow cocoon strains (T(W;2)Y-Chu, -Abe and -Ban types) from different Japanese stock centers. Of 12 RAPD markers identified in the normal W chromosomes of most silkworm strains in Japan, the newly irradiated W(B-YL-YS)Ze chromosome contained three, the T(W;2)Y-Chu chromosome contained six, and the T(W;2)Y-Abe and -Ban chromosomes contained only one (W-Rikishi). To investigate the ability of the reduced W-chromosome translocation fragments to form heterochromatin bodies, which are found in nuclei of normal adult female sucking stomachs, we examined cells of the normal type p50 strain and the T(W;2)Y-Chu and -Abe strains. A single sex heterochromatin body was found in nuclei of p50 females, whereas we detected only small sex heterochromatin bodies in the T(W;2)Y-Chu strain and no sex heterochromatin body in the T(W;2)Y-Abe strain. Since adult females of all strains were normal and fertile, we conclude that only extremely limited region, containing the W-Rikishi RAPD sequence of the W chromosome, is required to determine femaleness. Based on a comparison of the normal W-chromosome and 7 translocation and W-deletion strains we present a map of Fem relative to the 12 W-specific RAPD markers.

Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5' promoter.

It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5' promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

The female-killing chromosome of the silkworm, Bombyx mori, was generated by translocation between the Z and W chromosomes.

Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome, Df(p ( Sa ) + ( p )W + ( od ))Fem, derived from the translocation-carrying W chromosome (p ( Sa ) + ( p )W + ( od )), is inert as femaleness determinant. Moreover, this Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome has been thought to have a female-killing factor because no female larvae having the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome are produced. Initially, to investigate whether the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome contains any region of the W chromosome or not, we analyzed the presence or absence of 12 W-specific RAPD markers. The Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome contained 3 of 12 W-specific RAPD markers. These results strongly indicate that the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome contains the region of the W chromosome. Moreover, by using phenotypic and molecular markers, we confirmed that the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome is connected with a partially deleted Z chromosome and that this fused chromosome behaves as a Z chromosome during male meiosis. Furthermore, we demonstrated that the ZZW-type triploid female having the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome is viable. Therefore, we concluded that the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome does not have a female-killing factor but that partial deletion of the Z chromosome causes the death of the ZW-type diploid female having the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome. Additionally, our results of detailed genetic analyses strongly indicate that the female-killing chromosome composed of the Df(p ( Sa ) + ( p )W + ( od ))Fem chromosome and deleted Z chromosome was generated by translocation between the Z chromosome and the translocation-carrying W chromosome, p ( Sa ) + ( p )W + ( od ).

Transcription factor specificity protein 1 (Sp1) is the main regulator of nerve growth factor-induced sphingosine kinase 1 gene expression of the rat pheochromocytoma cell line, PC12.

Sphingosine kinase (SPHK) is known to exert an anti-apoptic role in various cells and cell lines. We previously reported that human brain is rich in SPHK1 (Murate et al. 2001). After showing a high expression of SPHK1 in rat brain, we examined the gene expression mechanism using nerve growth factor (NGF)-stimulated rat PC12 cells. With RT-PCR, we found that both rat brain and PC12 utilized exon 1d mostly out of eight untranslated first exons. NGF induced an increase in SPHK enzyme activity and protein about double those in PC12 cells, and NGF-induced SPHK1 mRNA was three times higher than in the control. The minimal 5' promoter was determined, and TrkA specific inhibitor K252a inhibited the NGF-induced promoter activity of SPHK1. The truncation or mutation of putative transcription factor-binding motifs revealed that one specificity protein 1 (Sp1) binding motif of the 5' region of exon 1d is prerequisite. Electrophoresis mobility shift assay confirmed the promoter analysis, indicating increased Sp1 protein binding to this motif after NGF treatment. Chromatin immunoprecipitation assay also showed the binding of Sp1 and the promoter region in vivo. These results suggest the signal transduction pathway from NGF receptor TrkA to transcription factor Sp1 protein binding to the promoter Sp1-like motif in NGF-induced rat SPHK1 gene expression.

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

Implication of prostaglandin E(2) in TNF-alpha-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs.

OBJECTIVE: Calpains are known as Ca(2+)-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined. METHODS: Human chondrocytic HCS-2/8 cells were stimulated by TNF-alpha in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined. RESULTS: TNF-alpha (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E(2) (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha. CONCLUSIONS: These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process.

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis.

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.

Mutations of the silkworm molybdenum cofactor sulfurase gene, og, cause translucent larval skin.

Normal silkworms (Bombyx mori) have opaque larval skin due to uric acid accumulation in the epidermis while a mutant, og, is translucent owing to a deficiency in xanthine dehydrogenase (XDH), which synthesizes uric acid. Molybdenum cofactor (MoCo) sulfurase is responsible for XDH activation in various organisms. A silkworm MoCo sulfurase gene was cloned and found to be on the og locus, whose mutant alleles, og(k) and og(t), show premature stop codons, proving that og is the MoCo sulfurase gene. It was observed that a miniature inverted-repeat transposable element (MITE), named Organdy, when inserted in an og(t) mutant allele exon, causes unstable splicing of a downstream intron leading to incomplete open reading frames.

Gelsolin suppresses tumorigenicity through inhibiting PKC activation in a human lung cancer cell line, PC10.

Gelsolin expression is frequently downregulated in lung cancer and several types of different human cancers. To examine the effects of gelsolin restoration on tumorigenicity, we here stably expressed various levels of gelsolin via gene transfer in lung cancer cells (squamous cell carcinoma line, PC10). We observed the alterations in tumorigenicity in vivo when implanted in nude mice, and the changes in growth properties in vitro. As compared to parental cells and control clones, gelsolin transfectants highly reduced tumorigenicity and repressed cell proliferation. Moreover, we investigated bradykinin-induced responses in gelsolin-overexpressing clones, because agonist-stimulated activation of the phospholipases C (PLC)/protein kinase C (PKC) signal transduction pathway is critical for cell growth and tumorigenicity. Bradykinin promotes phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by PLC and translocation of various PKC isoforms from the cytosolic fraction to the particulate fraction. Bradykinin treatment did not increase inositoltriphosphate (IP3) production and induce the membrane fractions of PKC alpha and PKC gamma in gelsolin tranfectants, while it induced PIP2 hydrolysis and increased the fractions in parental and control clones. These results suggest that gelsolin suppressed the activation of PKCs involved in phospholipid signalling pathways, inhibiting cell proliferation and tumorigenicity.

Identification of three annexin IX isoforms generated by alternative splicing of the carboxyl-terminal exon in silkworm, Bombyx mori.

Annexins (ANXs) are a family of structurally related proteins with Ca(2+)-dependent phospholipid-binding properties. Here we report the cloning of three cDNAs each encoding annexin IX (ANX IX) isoforms from unfertilized eggs of the silkworm, Bombyx mori. The analysis of exon/intron structures showed that the three mRNAs, named ANX IX-A (2300bp), ANX IX-B (1884bp) and ANX IX-C (1409bp), respectively, were generated from a single gene by alternative usage of a 3'-splice site of the last exon. Thus the three isoforms have an identical sequence from amino acid residues 1 to 307 and this region shows approximately 77% identity to Drosophila melanogaster ANX IX. Only amino acid residues 308-324 (A) or 308-323 (B and C), which correspond to the C-terminal tail, are different in the three proteins. A RT-PCR analysis indicated that the three isoforms of silkworm ANX IX were specifically expressed in various larval tissues and development stages. Interestingly, the C-terminal tail in ANXs I, II and V were previously confirmed as a binding region for protein kinase C. Thus generation of the three ANX IX isoforms in the silkworm, that are different from other ANXs, may have a functional significance other than binding to Ca(2+).

Caspase activation during hepatocyte apoptosis induced by tumor necrosis factor-alpha in galactosamine-sensitized mice.

BACKGROUND/AIMS: To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor-alpha (TNF-alpha), caspase cascade and ceramide formation were investigated in the liver of D-galactosamine (GalN)-sensitized mice treated with TNF-alpha. METHODS: Seven-week-old male BALB/c mice were intraperitoneally injected with 20 mg GalN 30 min prior to the intravenous injection of recombinant mouse TNF-alpha (0.5 microg/mouse). Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using chromogenic peptides as substrates. Ceramide content was determined using Escherichia coli diacylglycerol kinase. RESULTS: Apoptosis of hepatocytes was observed in mice treated with both GalN and TNF-alpha (GalN/TNF-alpha), but not GalN or TNF-alpha alone. Activation of caspases-9 and -3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF-alpha. In a cell-free system, processing of procaspases-9 and -3, and cytochrome c release were observed in the postnuclear fraction of liver obtained from GalN/TNF-alpha-treated mice, but not in that from control mice. Processing of procaspase-3 was inhibited by a caspase-9 inhibitor, but not by inhibitor for caspase-8 or -2. In a reconstitution assay system, procaspase-9 processing occurred, when both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF-alpha. Ceramide accumulation was observed only in apoptotic liver and preceded cytochrome c release and caspase activation. CONCLUSION: Cytochrome c release and caspase-9 activation are required for the activation of executor caspase-3 in TNF-alpha-induced hepatocyte apoptosis, but caspases-8 and -2 play, if any, a minimal role. Ceramide may be implicated in this apoptotic process.