Mode-selective optical packet switching in mode-division multiplexing networks.

A novel mode-selective optical packet switching, based on mode-multiplexers/demultiplexers and multi-port optical micro-electro-mechanical systems (MEMS) switches, has been proposed and experimentally demonstrated. The experimental demonstration was performed using the LP(01), LP(11a) and LP(11b) modes of a 30-km long mode-division multiplexed few-mode fiber link, utilizing 40 Gb/s, 16-QAM signals.

Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin.

The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.

Hypoxia selects for high-metastatic Lewis lung carcinoma cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors.

Low oxygen tension (hypoxia) is a common feature of solid tumors and stimulates the expressions of a variety of genes including those related to angiogenesis, apoptosis and endoplasmic reticulum (ER) stress response. Here we show a close correlation between metastatic potential and the resistance to hypoxia- and ER stress-induced apoptosis among the cell lines with differing metastatic potential derived from Lewis lung carcinoma. An apoptosis-specific expression profiling and immunoblot analyses revealed that the expression of antiapoptotic Mcl-1 increased as the resistance to apoptosis increased. Downregulation of the Mcl-1 expression in the high-metastatic cells by Mcl-1 small interfering RNA increased the sensitivity to hypoxia-induced apoptosis and decreased the metastatic ability. The hypoxia-induced apoptosis was not associated with p53 accumulation, although at present it is not possible to conclude that apoptosis-induced apoptosis is p53-independent. There was no correlation between the expression levels of ER stress-response proteins GADD153, GRP78 and ORP150 and the resistance to hypoxia or ER stresses. In vitro, small numbers of the high-metastatic cells overtook the low-metastatic cells after exposure to several rounds of hypoxia and reoxygenation. In solid tumors initially established from equal mixtures, the proportion of the high-metastatic cells to low-metastatic cells was significantly higher in hypoxic areas. Moreover, the high-metastatic cells were overtaking the low-metastatic cells in some of the tumors. Thus, tumor hypoxia and ER stress may provide a physiological selective pressure for the expansion of the high-metastatic cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors.

Differential expression of a tropomyosin isoform in low- and high-metastatic Lewis lung carcinoma cells.

Two-dimensional electrophoretograms of newly synthesized polypeptides from low-metastatic (P29) and high-metastatic (D6) Lewis lung carcinoma cells were compared. The results showed that the synthesis of tropomyosin 2 (TM2) was significantly less in D6 cells than in P29 cells. Furthermore, suppression of TM2 synthesis was induced in P29 cells during incubation in medium containing dimethyl sulfoxide or butyric acid, which induced the metastatic phenotype of P29 cells. These results suggest that the suppression of TM2 synthesis is linked to the metastatic potential of Lewis lung carcinoma cells.

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.

Isolation of cDNA clones for mouse cytoskeletal gamma-actin and differential expression of cytoskeletal actin mRNAs in mouse cells.

We described the structures of mouse cytoskeletal gamma-actin cDNA clones and showed that there is strong conservation of the untranslated regions with human gamma-actin cDNA. In addition, we found that the expression levels of beta- and gamma-actin mRNAs are differentially controlled in various mouse tissues and cell types but are coordinately increased in the cellular growing state. These results suggest that there are multiple regulatory mechanisms of cytoskeletal actin genes and are consistent with the argument that beta- and gamma-actins might have functional diversity in mammalian cells.

Effect of butyric acid on lung-colonizing ability of cloned low-metastatic Lewis lung carcinoma cells.

The lung-colonizing ability of low-metastatic Lewis lung carcinoma cells (P-29) was enhanced by their in vitro treatment with butyric acid and its sodium salt, sodium butyrate. Of the short chain fatty acids tested, butyric acid was the most effective in enhancing the lung-colonizing ability of P-29 cells; propionic acid and valeric acid were slightly effective, but acetic acid and caproic acid were ineffective. The enhancing effect of butyric acid on the lung-colonizing ability of P-29 cells was reversible, indicating that the result was the consequence of epigenetic alterations. Treatment of P-29 cells with butyric acid resulted in enhancement of secretion of plasminogen activator, cellular cathepsin B activity, and cellular adhesiveness. The phenotypes of cells treated with butyric acid were compared with those of cells treated with dimethyl sulfoxide, which was reported to enhance the lung-colonizing ability of P-29 cells. Significant differences were found in the phenotypes, especially that of cellular adhesiveness; that is, butyric acid enhanced mainly homotypic aggregation of the cells, while dimethyl sulfoxide enhanced mainly heterotypic adhesion, such as adhesion to monolayers of endothelial cells. In addition, butyric acid reversibly caused hyperacetylation of core histones in P-29 cells, while dimethyl sulfoxide did not.

Enhanced metastatic potential of cloned low-metastatic Lewis lung carcinoma cells treated in vitro with dimethyl sulfoxide.

Treatment of low-metastatic Lewis lung carcinoma cells (P-29) with dimethyl sulfoxide in vitro enhanced their lung-colonizing ability. The concentration of dimethyl sulfoxide used delayed the in vitro growth of P-29 cells but was not cytotoxic. The arrest and retention in the lung of untreated and dimethyl sulfoxide-treated P-29 cells labeled with 5-[125I]iodo-2'-deoxyuridine after injecting them into a tail vein of syngeneic mice were examined. Dimethyl sulfoxide-treated P-29 cells were trapped in the lung more than untreated cells and were cleared from the lungs more slowly than untreated cells. Treatment of P-29 cells with dimethyl sulfoxide resulted in the increase in their homotypic aggregation and adhesion to plastic culture dishes, monolayers of endothelial cells, and a subendothelial extracellular matrix. This treatment also increased significantly their activities of degradative enzymes, such as glycosidases and cathepsin B, and their production of plasminogen activator. These results indicate that the enhanced lung-colonizing ability of P-29 cells treated with dimethyl sulfoxide is due to the increase in adhesiveness, resulting in arrest and retention of the cells in the lung of the host and in the increase in their degradative enzyme activities. The enhancing effect of dimethyl sulfoxide on the lung-colonizing ability of P-29 cells was found to be reversible.

Effects of 12-O-tetradecanoylphorbol-13-acetate on adhesiveness and lung-colonizing ability of Lewis lung carcinoma cells.

The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the adherence of low-metastatic Lewis lung carcinoma cells (P-29) to the surface of plastic culture dishes and to monolayers of endothelial cells. This effect was transient, being apparent within 15 min and maximal within 1 h after treatment with TPA. Biologically active analogues of TPA and mezerein also enhanced attachment of P-29 cells, whereas inactive analogues of TPA did not. TPA-treated P-29 cells formed many more pulmonary nodules than did untreated P-29 cells when injected i.v. into C57BL/6 mice. The kinetics of enhancement of attachment of P-29 cells after TPA treatment coincided well with that of enhancement of their lung-colonizing ability. Addition of TPA to P-29 cells stimulated phosphorylation of a cellular protein with a molecular weight of 54,000. The possibility that this phosphorylation was related to activation of Ca2+ phospholipid-dependent protein kinase was suggested by the fact that phospholipid breakdown induced by exogenous treatment of the cells with Clostridium perfringens phospholipase C and 1-oleoyl-2-acetylglycerol also enhanced Mr 54,000 cellular protein phosphorylation. However, neither phospholipase C nor 1-oleoyl-2-acetylglycerol enhanced attachment of P-29 cells or their lung-colonizing ability.

Effects of retinoids on induction of differentiation of cultured mouse myeloid leukemia cells.

Retinoic acid, retinol, retinyl acetate, and retinal induced activities of lysosomal enzymes, such as lysozyme, acid protease, and acid phosphatase, in mouse myeloid leukemia cells (M1), while the pyridyl analog of retinoic acid had no effect. Retinoic acid was the most potent inducer of lysosomal enzyme activities. The induction of lysozyme activity by retinoic acid was inhibited by treatment with puromycin. The retinoids did not induce phagocytic and locomotive activities or morphological changes in M1 cells, and they inhibited the induction of these differentiation-associated properties by various inducers without inhibiting cell growth. Retinoic acid was the most potent inhibitor of induction of these differentiation-associated properties. The inhibitory effect of retinoic acid was found to be reversible. These results suggest that distinct mechanisms exist for control of induction of lysosomal enzyme activities and of other differentiation-associated properties of M1 cells, such as phagocytosis, morphological changes, and migration.

Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells.

We examined whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the promoter of the vascular endothelial growth factor (VEGF) gene that is activated by hypoxia is effective in killing highly metastatic Lewis lung carcinoma A11 cells under hypoxic conditions. We isolated the promoter region encompassing the hypoxia response element (HRE) of the mouse VEGF gene. To assess the hypoxia responsiveness of the VEGF promoter, A11 cells were transiently transfected with luciferase reporter plasmids. Exposure of the transfectants to hypoxia resulted in a 2-3-fold induction of luciferase activity. Deletion of the HRE site abolished VEGF promoter activity under both normoxic and hypoxic conditions. We constructed a retroviral vector harboring the HSV-TK or green fluorescence protein (GFP) gene under the control of the VEGF promoter. A11 cells transfected with vector harboring the VEGF promoter fused to the HSV-TK gene [A11(HRE/TK) cells] were more sensitive to ganciclovir than cells transfected with the control vector harboring the VEGF promoter alone, and the sensitivity of the A11(HRE/TK) cells was increased by exposure to hypoxia followed by reoxygenation. Culturing A11 cells transfected with vector harboring the VEGF promoter fused to the GFP gene under hypoxic conditions resulted in an increase in the expression of GFP. Monitoring GFP expression and vascularity in the A11 transfectant tumors revealed up-regulation of GFP expression in poorly vascularized regions. Administration of ganciclovir to mice bearing s.c. tumors formed by A11(HRE/TK) cells resulted in regression of the tumors. These results suggest a possible application of the suicide gene driven by the VEGF promoter to cancer gene therapy that efficiently targets hypoxic tumor cells.

Production of differentiation-inhibiting factor in cultured mouse myeloid leukemia cells treated with retinoic acid.

Mouse myeloid leukemia cells (M1) could be induced to differentiate into mature macrophages and granulocytes with dexamethasone or proteinaceous inducer. Retinoic acid inhibited functional and morphological differentiation of M1 cells, but the pyridyl analog of retinoic acid had no effect. M1 cells could be induced to produce a factor(s) inhibiting their own differentiation to macrophage- and granulocyte-like cells by retinoic acid but not by its pyridyl analog. This factor(s) inhibited induction by inducers of phagocytic activity, locomotive activity, lysozyme activity, and morphological changes in M1 cells. The production of the inhibitory factor(s) by M1 cells incubated with retinoic acid was inhibited by a low concentrations (5--10 ng/ml) of actinomycin D. The inhibitory factor seemed to be a protein(s), since it was susceptible to heat treatment and proteases. The effect of retinoic acid in inducing production of the inhibitory factor(s) by M1 cells seemed to be reversible, since it was low on washing the cells with fresh medium. Therefore, induction of this inhibitory factor may be involved in the mechanism of inhibition of functional and morphological differentiation of M1 cells by retinoic acid.

Characterization of factors stimulating differentiation of mouse myeloid leukemia cells from a Yoshida sarcoma cell line cultured in serum-free medium.

A clone, YS-T22, of cells from Yoshida sarcoma cell line, YSSF-212T, grown in "serum-free" culture medium produced factors stimulating differentiation of mouse myeloid leukemia cells (M1) to macrophages and granulocytes. The formation of macrophages and granulocytes was accompanied by induction of phagocytosis, locomotive activity, and lysosomal enzyme activities. The rates of induction of these differentiated phenotypes were proportional to the concentration of the factor added and the period of treatment. The factor stimulating differentiation of M1 cells was a heat-labile, nondialyzable proteinaceous substance that was inactivated by trypsin but not by ribonuclease or glycosidases. On diethylaminoethyl cellulose chromatography, the factor stimulating differentiation of M1 cells from conditioned medium of YS-T22 cells was eluted in various fractions with or without activity of the colony-stimulating factor.

Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer.

The proinflammatory interleukin-33 (IL-33) binds to its receptor ST2L on the surface of immune cells and stimulates the production of Th2 cytokines; however, the effects of IL-33 on tumour cells are poorly understood. Here we show that ST2 was significantly downregulated in human lung cancer tissues and cells compared with normal lung tissues and cells. IL-33 expression was also inversely correlated with the stages of human lung cancers. In accordance with this finding, low-metastatic cells but not high-metastatic cells derived from Lewis lung carcinoma expressed functional ST2L. IL-33 was abundantly present in the tumours established by the low-metastatic cells compared with those formed by the high-metastatic cells. Although the low-metastatic cells scarcely expressed IL-33 in vitro, these cells did expry 6ess this molecule in vivo, likely due to stimulation by intratumoural IL-1ß and IL-33. Importantly, IL-33 enhanced the cell death of ST2L-positive low-metastatic cells, but not of ST2L-negative high-metastatic cells, under glucose-depleted, glutamine-depleted and hypoxic conditions through p38 MAPK and mTOR activation, and in a mitochondria-dependent manner. The cell death was characterised by cytoplasmic blisters and karyolysis, which are unique morphological features of oncosis. Inevitably, the low-metastatic cells, but not of the high-metastatic cells, grew faster in IL-33(-/-) mice than in wild-type mice. Furthermore, IL-33 selected for the ST2L-positive, oncosis-resistant high-metastatic cells under conditions mimicking the tumour microenvironment. These data suggest that IL-33 enhances lung cancer progression by selecting for more malignant cells in the tumour microenvironment.

Expression of antisense RNA to S100A4 gene encoding an S100-related calcium-binding protein suppresses metastatic potential of high-metastatic Lewis lung carcinoma cells.

S100A4 (also known as pEL98/mts1/p9Ka/18A2/42A/calvasculin /FSP1/CAPL), a member of S100-related calcium-binding proteins, has been implicated to play a role in metastasis. In the present study, we examined the effect of antisense S100A4 RNA on metastatic potential of Lewis lung carcinoma (LLC) cells. High-metastatic All cells were transfected with the expression vector containing S100A4 cDNA in an inverted (antisense) orientation under the transcriptional control of the mouse metallothionein promoter. Treatment of a stably transfected clone (AS10 cells) with Zn2+ resulted in the suppression of the experimental metastatic ability, which was accompanied with the expression of antisense S100A4 RNA and the suppression of the S100A4 expression at both the mRNA and the protein levels. To further confirm the effect of antisense S100A4 RNA, we established several clones after retroviral transduction with an antisense S100A4 construct. Notably, reduced metastatic potential was also evident in these clones. In the antisense S100A4 RNA-expressing cells, cell motility and in vitro invasiveness were found to be suppressed.

Role of a signal transduction pathway which controls disassembly of microfilament bundles and suppression of high-molecular-weight tropomyosin expression in oncogenic transformation of NRK cells.

Role of disassembly of microfilament bundles and suppression of high-molecular-weight tropomyosin (TM) expression in growth factor- and various oncogene-induced transformation was studied by using NRK cells and its transformation-deficient mutants. In NRK cells which show a transformed phenotype by treatment with EGF and TGF-beta, cellular stress fibers became dissociated by EGF or EGF and TGF-beta combination, whereas TGF-beta alone caused thicker appearance of stress fibers. Accompanying these changes, the expression of TM isoforms 1 and 2 was suppressed by treatment with EGF or EGF and TGF-beta, but elevated by TGF-beta with similar time courses. On the other hand, the transformation-deficient mutant cell lines, 39-1 and 39-3, did not show the transformed phenotypes by treatment with EGF and TGF-beta. Neither EGF nor EGF and TGF-beta combination affected cellular stress fibers and expression of TM isoforms 1 and 2 in both mutant lines. The relationship between the formation of stress fibers and the expression of TM isoforms was consistent in NRK cells, the mutant lines and their various oncogene-expressing sublines under various culture conditions. NRK cells overexpressing exogenous mouse TM isoform 2 showed markedly decreased susceptibility to EGF-induced dissociation of stress fibers and decreased anchorage-independent growth potential in the presence of EGF and TGF-beta. These results indicate that the transformation-deficient NRK mutant lines, 39-1 and 39-3 have defects in an EGF signal transduction pathway which induces suppression of high-molecular-weight TM expression and disassembly of microfilament bundles and suggested that the activation of the pathway is important for morphological transformation and oncogenic growth in growth factors- and various oncogene-induced transformation of NRK cells.

Nucleotide sequence of cDNA for nonmuscle tropomyosin 5 of mouse fibroblast.

A cDNA clone for mouse fibroblast tropomyosin (TM) 5 was obtained from a cDNA library using human TM pseudogene as a probe. Sequence analysis of the clone revealed that the deduced amino acid sequence is different only at the 4th position from the human counterpart and that both the 5' and 3' untranslated regions are highly conserved.

Increased expression of S100A4, a metastasis-associated gene, in human colorectal adenocarcinomas.

The S100A4 gene (also known as pEL98/mts1/p9Ka/18A2/42A/calvasculin /FSP1/CAPL) encoding an S100-related calcium-binding protein is implied to be involved in the invasion and metastasis of murine tumor cells. In the present study, the expression of S100A4 in human colorectal adenocarcinoma cell lines (SW837, LoVo, DLD-1, HT-29, SW480, SW620, WiDr, and Colo201) and surgically resected neoplastic tissues was examined to investigate whether S100A4 plays a role in the invasion and metastasis of human tumor cells. Northern blot analysis using total RNA isolated from the adenocarcinoma cell lines revealed that five of the eight cell lines expressed substantial amounts of S100A4 mRNA. Normal colon fibroblasts (CCD-18Co) expressed little of the RNA. Using surgically resected specimens, it seemed that the amount of S100A4 mRNA in adenomas was nearly equal to that in normal colonic mucosa, whereas adenocarcinomas expressed a significantly higher amount of the RNA than did the adjacent normal colonic mucosa. Immunohistochemical analysis using formalin-fixed paraffin-embedded surgical specimens and monoclonal anti-S100A4 antibody demonstrated that none of 12 adenoma specimens were immunopositive, whereas 8 of 18 (44%) focal carcinomas in carcinoma in adenoma specimens and 50 of 53 (94%) adenocarcinoma specimens were immunopositive. Interestingly, the incidence of immunopositive cells increased according to the depth of invasion, and nearly all of the carcinoma cells in 14 metastases in the liver were positive. These results suggest that S100A4 may be involved in the progression and the metastatic process of human colorectal neoplastic cells.

Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity.

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5'-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with the UPRT gene (Colon 26/UPRT cells) and tested the in vivo antitumoral effect of 5-FU in syngeneic immunocompetent mice. After 5-FU administration, tumors of Colon 26/UPRT cells regressed, whereas those of wild-type cells were unaffected. The mice that once eliminated Colon 26/UPRT tumors after 5-FU treatment rejected wild-type cells that were subsequently inoculated but not irrelevant syngeneic tumor cells. This suicide gene/prodrug system was less efficient in nude mice, suggesting that mature alphabeta T cells play a role in the antitumoral effect. The cytotoxicity mediated by the bystander effect was marginal in this system, contrary to the herpes simplex virus-thymidine kinase gene/ganciclovir system. Therefore, expression of the UPRT gene in tumor cells followed by 5-FU administration is a possible strategy for cancer gene therapy, but potentiation of the bystander effect is required for its therapeutic application.

Induced immunity by expression of interleukin-2 or GM-CSF gene in murine neuroblastoma cells can generate antitumor response to established tumors.

We examined whether antitumor immunity could be generated by the inoculation of cytokine-producing murine neuroblastoma cells (C1300), and whether the immunity might be effective for the established tumors of wild-type (wt) cells. For that purpose, we transduced low immunogenic C1300 cells with interleukin-2 (IL-2), GM-CSF, or IL-4 genes. A loss of tumorigenicity in syngeneic mice was observed using IL-2- and GM-CSF- but not IL-4-producing C1300 cells, although their in vitro growth rates were not affected by the transduction. The syngeneic mice that had rejected IL-2 or GM-CSF producers did not develop tumors of wt cells inoculated subsequently, but formed tumors of irrelevant syngeneic mammary tumor cells. Accordingly, the inoculation of IL-2 or GM-CSF producers into immunocompetent mice generated tumor-specific acquired immunity. The induced immunity using IL-2 or GM-CSF producers was also effective in eradicating established subcutaneous tumors of wt cells and in reducing the number of preexisting metastatic foci in the liver. These data suggest a potential application of IL-2- or GM-CSF-producing syngeneic tumor cells for the treatment of low immunogenic neuroblastomas.