BBA - Molecular Cell Research (v.1743, #1-2)

ERK2 is required for FGF1-induced JNK1 phosphorylation in Xenopus oocyte expressing FGF receptor 1 by Edith Browaeys-Poly; Véronique Fafeur; Jean Pierre Vilain; Katia Cailliau (1-4).
A possible connection between the ERK2 and JNK1 MAP kinases transduction cascades was investigated in Xenopus oocytes expressing FGFR1 stimulated by FGF1. Injection of various inhibitors for the Shc/Grb2/Ras/Mos/MEK/ERK2 cascade blocked FGF1-induced germinal vesicle breakdown (GVBD), as well as ERK2 and JNK1 phosphorylation. JNK1 was found to be activated downstream of ERK2, since injection of an active ERK2 triggered JNK1 phosphorylation and inhibition of ERK2 either by a MEK inhibitor or the MKP3 phosphatase blocked JNK1 phosphorylation. These results demonstrated that in FGFR1 signalling JNK1 phosphorylation depends on ERK2.
Keywords: Fibroblast growth factor receptor; JNK; ERK; Xenopus oocyte;

The plastids of many algae are surrounded by three or four membranes, thought to be a consequence of their evolutionary origin through secondary endosymbiosis between photosynthetic and non-photosynthetic eukaryotes. Each membrane constitutes a barrier to the passage of proteins, so protein targeting in these complex plastids has an extra level of difficulty when compared to higher plants. In the latter, protein translocation across the two membranes uses multi-protein complexes that together import proteins possessing an N-terminal leader sequence rich in serine and threonine (S/T). In contrast, while targeting to most complex plastids also involves an S/T-rich region, this region is preceded by an N-terminal hydrophobic signal peptide. This arrangement of peptide sequences suggests that proteins directed to complex plastids pass through the ER, as do other proteins with hydrophobic signal peptides. However, this simplistic view is not always easy to reconcile with what is known about the different secondary plastids. In the first group, with plastids bounded by three membranes, plastid-directed proteins do indeed arrive in Golgi-derived vesicles, but a second hydrophobic region follows the S/T-rich region in all leaders. In the second group, where four membranes completely surround the plastids, it is still not known how the proteins arrive at the plastids, and in addition, one member of this group uses a targeting signal rich in asparagine and lysine in place of the S/T-rich region. In the third group, the fourth bounding membrane is contiguous with the ER, but it is not clear what distinguishes plastid membranes from others in the endomembrane system. Knowing what to expect is important, as genomic sequencing programs may soon be turning up some of the missing pieces in these translocation puzzles.
Keywords: Secondary chloroplast; Protein targeting; Signal peptide; Transit peptide;

Heat shock proteins (hsps) play an important role in maintaining cellular homeostasis and protecting cells from various insults. Recent evidence also implicates hsps in the regulation of the immune response, particularly the inflammatory process. In the present study, we showed that human promonocytic cells (THP-1) produced elevated levels of tumor necrosis factor alpha (TNFα) after incubation with bacterial lipopolysaccharide (LPS) when cells were pre-stressed by a mild heat shock (HS) of 42 °C (1.5 h) followed by recovery at 37 °C (3 h) in comparison with non-stressed cells also stimulated with LPS. This enhanced TNFα production was not due to changes in nuclear factor-κB (NF-κB) activation, TNFα transcription rates, or mRNA stability. Thus, an effect at the translational or posttranslational level is likely responsible. Elevated production of TNFα was not observed when cells were stimulated with LPS immediately after stress or when HS temperature was increased to 43 °C. This negative effect of HS is likely due to a harmful effect of temperature. Moreover, enhanced LPS-induced TNFα production was not observed after differentiation of promonocytes into macrophage-like cells. Thus, our results show that the stress temperature, recovery period, and differentiation stage of the cell modulate the effect of HS on the inflammatory process.
Keywords: Inflammation; Lipopolysaccharide; Macrophage; Heat shock proteins; Stress;

Erythropoietin (Epo) promotes the development of erythroid progenitors by triggering intracellular signals through the binding to its specific receptor (EpoR). Previous results related to the action of aluminum (Al) on erythropoiesis let us suggest that the metal affects Epo interaction with its target cells. In order to investigate this effect on cell activation by the Epo–EpoR complex, two human cell lines with different dependence on Epo were subjected to Al exposure. In the Epo-independent K562 cells, Al inhibited Epo antiapoptotic action and triggered a simultaneous decrease in protein and mRNA EpoR levels. On the other hand, proliferation of the strongly Epo-dependent UT-7 cells was enhanced by long-term Al treatment, in agreement with the upregulation of EpoR expression during Epo starvation. Results provide some clues to the way by which Epo supports cell survival and growth, and demonstrate that not all the intracellular factors needed to guarantee the different signaling pathways of Epo-cell activation are available or activated in cells expressing EpoR. This study then suggests that at least one of the mechanisms by which Al interfere with erythropoiesis might involve EpoR modulation.
Keywords: Erythropoietin; Aluminum; Erythropoietin receptor; K562 cell; UT-7 cell line; Apoptosis;

Contact of Chlamydophila pneumoniae with type II cell triggers activation of calcium-mediated NF-κB pathway by Heide Wissel; Torsten Müller; Mario Rüdiger; Matthias Krüll; Roland R. Wauer (37-48).
Nuclear factor-κB (NF-κB) plays an important role in inflammation, proliferation and regulation of apoptosis. The purpose of the present study on type II cells was to investigate whether Chlamydophila pneumoniae contact induces (I) a Ca2+ release, that (II) disrupts F-actin/β-tubulin cytoskeletal association with NF-κB/IκBα, leading to (III) a subsequent NF-κB activation.Incubation of rat type II pneumocytes with C. pneumoniae caused an intracellular calcium release within seconds. Confocal laser scanning microscopy (CLSM) revealed that bacterial contact with cell surface leads to a disappearance of the microvilli and disturbs the co-localization between F-actin and NF-κB (p65). Using semi-quantitative CLSM, we show that at 10–30 min IκBα was decreased and p65 or p50 was simultaneously translocated from cytoplasm to the nucleus, resulting in a 19-fold and 17-fold increase versus control cells. During this time no bacteria were internalized into type II cells. The pre-treatment of cells with BAPTA-AM inhibited C. pneumoniae-mediated calcium release. BAPTA-AM or SN50 prevented the C. pneumoniae-induced changes in F-actin cytoskeleton and inhibited NF-κB activation. Paclitaxel reduced C. pneumoniae-mediated changes of β-tubulin cytoskeleton and activation of NF-κB. These results suggest that calcium-mediated cytoskeleton reorganization is involved in C. pneumoniae-induced NF-κB activation in type II cells.
Keywords: Chlamydophila pneumoniae; Calcium; Cytoskeleton; NF-κB;

Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid by Yoichiro Takami; Itaru Yamamoto; Hirohito Tsubouchi; Eiichi Gohda (49-56).
Topical treatment of skin with all-trans-retinoic acid (ATRA), the major biologically active form of vitamin A, results in hyperproliferation of basal keratinocytes, leading to an accelerated turnover of epidermis cells and thickening of the epidermis, probably via induction of production of paracrine growth factors for keratinocytes in epidermal suprabasal keratinocytes and/or dermal fibroblasts. Since hepatocyte growth factor (HGF) is a factor mitogenic to epidermal keratinocytes secreted from dermal fibroblasts, the effect of ATRA on basal and induced HGF production in human dermal fibroblasts in culture was examined. ATRA alone did not induce HGF production, but it significantly enhanced HGF production induced by the cAMP-elevating agent cholera toxin or the membrane-permeable cAMP analog 8-bromo-cAMP. Cholera toxin-induced activation of cAMP responsive element (CRE)-binding protein (CREB) was enhanced by pretreating cells with ATRA for 24 h. In contrast, HGF production induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) was potently inhibited by ATRA. These modulatory effects of ATRA were different from the effects of transforming growth factor-β1 (TGF-β) and dexamethasone, both of which inhibited HGF production induced by all of the four inducers. Up-regulation of HGF gene expression by cholera toxin and EGF was also enhanced and inhibited, respectively, by ATRA. Both 9-cis-retinoic acid (9-cis-RA) and 13-cis-retinoic acid (13-cis-RA), which are stereo-isomers of ATRA, showed a modulatory effect on HGF induction similar to that of ATRA. These results suggest that ATRA augments the induction of HGF production caused by increased intracellular cAMP.
Keywords: Hepatocyte growth factor; All-trans-retinoic acid; cAMP; Induction; Skin fibroblast; Keratinocyte;

The metabolism of hyaluronan in cultured rabbit growth plate chondrocytes during differentiation by A. Suzuki; K. Tanimoto; S. Ohno; Y. Nakatani; K. Honda; N. Tanaka; T. Doi; M. Ohno-Nakahara; K. Yoneno; M. Ueki; K. Tanne (57-63).
Hyaluronan (HA) is one of the major extracellular matrix components in cartilage. In addition to the biomechanical functions, HA has various important roles in the differentiation of chondrocytes. The purpose of this study was to clarify the nature of HA synthesis during chondrocyte differentiation. Growth plate chondrocytes were isolated from rabbit ribs and cultured in chondrocyte differentiation medium. The amount of HA and HA synthase (HAS) mRNA levels were analyzed for each stage of chondrocyte differentiation by means of high-performance liquid chromatography (HPLC) and real-time PCR, respectively. The distribution of HA in cultured chondrocytes was observed by histochemical staining. The amount of HA, ranging widely in size, was increased substantially during the hypertrophic stage. The expression levels of HAS2 and HAS3 mRNAs were low during the matrix-forming stage. HAS2 mRNA level was substantially enhanced at the pre-hypertrophic stage, whereas HAS3 mRNA level exhibited a slight increase. HAS1 mRNA was not detected. The intensity of HA staining was high around the hypertrophic chondrocytes. These results suggest that HA metabolism in chondrocyte differentiation is regulated by the selective expression of HASs, and HAS2 and the related large size-HA may have a certain association with the hypertrophic changes of chondrocytes
Keywords: Hyaluronan synthase; HAS; Chondrocyte; Differentiation;

Mechanism of DNA binding and localized strand separation by Purα and comparison with Pur family member, Purβ by Margaret J. Wortman; Edward M. Johnson; Andrew D. Bergemann (64-78).
Purα is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Purα unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Purα are essential for both ss- and duplex DNA binding. Purα binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Purα since removal of Purα in the gel eliminates the series and since Purα binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Purα binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Purβ lacking the Purα C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Purα can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Purα.
Keywords: DNA unwinding; DNA binding; Telomere; Transcription; Replication; c-Myc;

Iron regulatory protein 2 (IRP2), a posttranscriptional regulator of iron metabolism, is subjected to iron-dependent degradation by the proteasome. Recent experiments proposed a mechanism involving 2-oxoglutarate-dependent oxygenases. Enzymes of this class, such as prolyl-4-hydroxylases, mediate the oxygen and iron-dependent degradation of the hypoxia inducible factor HIF-1α, which requires the E3 ubiquitin ligase activity of pVHL. Considering that the pathways for IRP2 and HIF-1α degradation share remarkable similarities, we investigated whether pVHL may also be involved in the degradation of IRP2. We show here that IRP2 can interact with pVHL in co-transfection/co-immunoprecipitation assays. Furthermore, pVHL is able to promote the ubiquitination and the decay of transfected IRP2. However, the iron-dependent degradation of endogenous IRP2 is not impaired in VHL-deficient cell lines, suggesting that pVHL is not a necessary component of this pathway.
Keywords: Iron regulatory protein; Iron-responsive element; Transferrin receptor; Ferritin;

Expression of catalase and glutathione peroxidase in renal insufficiency by Ram K. Sindhu; Ashkan Ehdaie; Farbod Farmand; Kanwaljit K. Dhaliwal; Tri Nguyen; Chang-De Zhan; Christian K. Roberts; Nosratola D. Vaziri (86-92).
Chronic renal failure (CRF) is associated with oxidative stress, the precise mechanism of which is yet to be elucidated. The present study was undertaken to investigate in renal insufficiency the expression of catalase and glutathione peroxidase, which play a critical role in antioxidant defense system by catalyzing detoxification of hydrogen peroxide (H2O2) and organic hydroperoxides. Rats were randomly assigned to the CRF (5/6 nephrectomized) and sham-operated control groups and observed for 6 weeks. Renal and thoracic aortic catalase and glutathione peroxidase protein abundance was measured by Western blotting. The enzyme activities in the renal and aortic extracts, hepatic glutathione levels, blood pressure and urinary nitric oxide metabolites (NOx) excretion were also measured. Blood pressure and urinary nitric oxide metabolite (NO x ) excretion were also measured. The CRF group showed a significant down-regulation of both immunodetectable catalase and glutathione peroxidase proteins in the remnant kidney. Catalase activity was also significantly decreased in the remnant kidney whereas glutathione peroxidase activity was not significantly affected. Furthermore, the protein abundance of catalase was unchanged whereas the enzyme activity was significantly decreased in the thoracic aorta of CRF animals compared to the sham-operated controls. By contrast, both the protein abundance and the enzyme activity of glutathione peroxidase were not significantly affected in the aorta of CRF animals compared to the sham-operated controls. This was coupled with marked arterial hypertension, significant reduction of hepatic glutathione levels and urinary NO x excretion pointing to increased inactivation and sequestration of NO by superoxide. These events point to the role of impaired antioxidant defense system in the pathogenesis of oxidative stress in CRF.
Keywords: Hypertension; Nitric oxide; Uremic toxin; Oxygen-free radical; Anemia; Cardiovascular disease; Lipid peroxidation; Hydrogen peroxide; Glutathione;

Stable transfection of Acanthamoeba castellanii by Zhihua Peng; Romaica Omaruddin; Erik Bateman (93-100).
A simple method for stable transfection of Acanthamoeba castellanii using plasmids which confer resistance to neomycin G418 is described. Expression of neomycin phosphotransferase is driven by the Acanthamoeba TBP gene promoter, and can be monitored by cell growth in the presence of neomycin G418 or by Western blot analysis. Transfected cells can be passaged in the same manner as control cells and can be induced to differentiate into cysts, in which form they maintain resistance to neomycin G418 for at least several weeks, although expression of neomycin phosphotransferase is repressed during encystment. Expression of EGFP or an HA-tagged EGFP–TBP fusion can be driven from the same plasmid, using an additional copy of the Acanthamoeba TBP gene promoter or a deletion mutant. The TBP–EGFP fusion is localized to the nucleus, except in a small proportion of presumptive pre-mitotic cells. EGFP expression can also be driven by the cyst-specific CSP21 gene promoter, which is completely repressed in growing cells but strongly induced in differentiating cells. Transfected cells maintain their phenotype for several weeks, even in the absence of neomycin G418, suggesting that transfected genes are stably integrated within the genome. These results demonstrate the utility of the neomycin resistance based plasmids for stable transfection of Acanthamoeba, and may assist a number of investigations.
Keywords: Acanthamoeba castellanii; Gene expression; Stable transfection; Gene promoter; Gene induction; Gene repression; Protein expression; Transcription; RNA polymerase II; TBP; EGFP;

Induction of murine erythroleukemia (MEL) cell differentiation is accompanied by transcriptional activation of globin genes and biosynthesis of hemoglobin. In this study, we observed cytoplasmic accumulation of relatively small RNAs of different size (150–600 nt) hybridized to α1 and βmajor globin DNA probes in MEL cells blocked to differentiate by hypomethylating agents (neplanocin A, 3-deazaneplanocin A and cycloleucine). These RNAs lack poly(A) tail and appear to be quite stable. Search within the 3′-end flanking sequences of βmajor globin gene revealed the presence of a B1 repeat element, several ATG initiation codons, a GATA-1 consensus sequence and sequences recognized by AP-1/NF-E2 and erythroid Krüppel-like factor (EKLF) transcription factors. These data taken together indicate that exposure of MEL cells to hypomethylating agents promotes accumulation of relatively small discrete RNA transcripts lacking poly(A) tail regardless of the presence or absence of inducer dimethylsulfoxide (DMSO). However, the relative steady-state level of small RNAs was comparatively higher in cells co-exposed to inducer and each one of the hypomethylating agents. Although the orientation of these RNAs has not been established as yet, the possibility these small poly(A) RNAs which are induced by hypomethylating agents may be involved in the blockade of MEL cell differentiation program is discussed.
Keywords: MEL; Cell; Differentiation; Hypomethylating agent; Inducer; Globin gene; Transcription; Neplanocin A; 3-Deazaneplanocin A; Cycloleucine; Silent DNA; DNA methylation; RNA methylation;

High hydrostatic pressure (HP) increases Hsp70 protein and mRNA levels by increasing the mRNA half-life without activation of HSF1 transcription factor. We investigated whether this change in gene expression requires Hsp90, previously shown to regulate hsp70 genes via HSF1. In HeLa cells, both HP and Hsp90 inhibitor geldanamycin (GA) up-regulated Hsp70 expression through mRNA stabilisation. GA, unlike HP, increased HSF1 activation. However, when exposures were used together a marked Hsp70 response was observed with mRNA stabilisation without coincidence of HSF1 activation. Our data suggests that Hsp90 is involved in hsp70 mRNA stabilisation and the HSF1 activation can be suppressed by high HP.
Keywords: Hydrostatic pressure; Heat shock protein; mRNA stabilisation; Geldanamycin;

50-Hz extremely low frequency electromagnetic fields enhance cell proliferation and DNA damage: possible involvement of a redox mechanism by Federica I. Wolf; Angela Torsello; Beatrice Tedesco; Silvia Fasanella; Alma Boninsegna; Marcello D'Ascenzo; Claudio Grassi; Gian Battista Azzena; Achille Cittadini (120-129).
HL-60 leukemia cells, Rat-1 fibroblasts and WI-38 diploid fibroblasts were exposed for 24–72 h to 0.5–1.0-mT 50-Hz extremely low frequency electromagnetic field (ELF-EMF). This treatment induced a dose-dependent increase in the proliferation rate of all cell types, namely about 30% increase of cell proliferation after 72-h exposure to 1.0 mT. This was accompanied by increased percentage of cells in the S-phase after 12- and 48-h exposure. The ability of ELF-EMF to induce DNA damage was also investigated by measuring DNA strand breaks. A dose-dependent increase in DNA damage was observed in all cell lines, with two peaks occurring at 24 and 72 h. A similar pattern of DNA damage was observed by measuring formation of 8-OHdG adducts. The effects of ELF-EMF on cell proliferation and DNA damage were prevented by pretreatment of cells with an antioxidant like α-tocopherol, suggesting that redox reactions were involved. Accordingly, Rat-1 fibroblasts that had been exposed to ELF-EMF for 3 or 24 h exhibited a significant increase in dichlorofluorescein-detectable reactive oxygen species, which was blunted by α-tocopherol pretreatment. Cells exposed to ELF-EMF and examined as early as 6 h after treatment initiation also exhibited modifications of NFκB-related proteins (p65-p50 and IκBα), which were suggestive of increased formation of p65-p50 or p65-p65 active forms, a process usually attributed to redox reactions. These results suggest that ELF-EMF influence proliferation and DNA damage in both normal and tumor cells through the action of free radical species. This information may be of value for appraising the pathophysiologic consequences of an exposure to ELF-EMF.
Keywords: 8-OHdG; Single strand breaks; Cell cycle; DCF; NFκB; IκB; α-Tocopherol;

Clinorotation prevents differentiation of rat myoblastic L6 cells in association with reduced NF-κB signaling by Katsuya Hirasaka; Takeshi Nikawa; Louis Yuge; Ibuki Ishihara; Akira Higashibata; Noriaki Ishioka; Atsuko Okubo; Takashi Miyashita; Naoto Suzue; Takayuki Ogawa; Motoko Oarada; Kyoichi Kishi (130-140).
In this study, we examined effects of the three-dimensional (3D)-clinorotation, a simulated-model of microgravity, on proliferation/differentiation of rat myoblastic L6 cells. Differentiation of L6 cells into myotubes was significantly disturbed in the 3D-clinorotation culture system, although the 3D-clinorotation had no effect on the proliferation. The 3D-clinorotation also suppressed the expression of myogenesis marker proteins, such as myogenin and myosin heavy chain (MHC), at the mRNA level. In association with this reduced differentiation, we found that the 3D-clinorotation prevented accumulation of ubiquitinated proteins, compared with non-rotation control cells. Based on these findings, we focused on the ubiquitin-dependent degradation of IκB, a myogenesis inhibitory protein, to clarify the mechanism of this impaired differentiation. A decline in the amount of IκB protein in L6 cells was significantly prevented by the rotation, while the amount of the protein in the non-rotated cells decreased along with the differentiation. Furthermore, the 3D-clinorotation reduced the NF-κB-binding activity in L6 cells and prevented the ubiquitination of IκB proteins in the IκB- and ubiquitin-expressing Cos7 cells. Other myogenic regulatory factors, such as deubiquitinases, cyclin E and oxygen, were not associated with the differentiation impaired by the clinorotation. Our present results suggest that simulated microgravity such as the 3D-clinorotation may disturb skeletal muscle cell differentiation, at least in part, by inhibiting the NF-κB pathway.
Keywords: 3D-clinorotation; Rat myoblastic L6 cell; Ubiquitination; NF-κB signaling; IκB;

Nucleolar localization of hepatic c-Myc: a potential mechanism for c-Myc regulation by Jennifer A. Sanders; Philip A. Gruppuso (141-150).
The c-myc proto-oncogene encodes a transcription factor that is involved in cell proliferation, growth, differentiation, and apoptosis. Previous studies on the regulation of hepatic c-myc have focused on control of its mRNA expression, which generally correlates with hepatocyte proliferation during both liver development and liver regeneration. However, Western blot analysis showed similar levels of hepatic c-Myc in fetal and adult liver. We therefore went on to examine the abundance and distribution of hepatic c-Myc. Immunofluorescence on adult rat liver cryosections showed that c-Myc was readily detectable, but that it was largely localized to the nucleolus. In contrast, proliferating fetal hepatocytes and adult hepatocytes from regenerating liver showed a diffuse nuclear pattern. Transient transfection of adult hepatocytes with full-length HA-Myc also revealed localization to the nucleolus. Western immunoblotting studies confirmed that immunoreactive c-Myc was present in nucleolar extracts isolated from adult liver. We speculate that the nucleolus may act to sequester c-Myc in quiescent hepatocytes while providing a pool of c-Myc that is readily available to reach its targets in the nucleus.
Keywords: c-Myc; Nucleolus; Liver; Hepatocyte; Liver Regeneration;

TGF-β1-mediated activations of c-Src and Rac1 modulate levels of cyclins and p27Kip1 CDK inhibitor in hepatoma cells replated on fibronectin by Hwang-Phill Kim; Tai-Young Kim; Mi-Sook Lee; Hyun-Soon Jong; Tae-You Kim; Jung Weon Lee; Yung-Jue Bang (151-161).
Integrin-mediated cell adhesion transduces signals to regulate actin cytoskeleton and cell proliferation. While understanding how integrin signals cross-talk with the TGF-β1 pathways, we observed lamellipodia formation and cyclin regulation in Hep3B cells, following TGF-β1 treatment. To answer if integrin signaling via actin organization might regulate cell cycle progression after TGF-β1 treatment, we analyzed cross-talk between the two receptor-mediated pathways in hepatoma cells on specific ECMs. We found that basal and TGF-β1-mediated activation of c-Src and Rac1, expression of cyclins E and A, and suppression of p27Kip1 were significant in cells replated on fibronectin, but not in cells on collagen I, indicating a different integrin-mediated cellular response to TGF-β1 treatment. Levels of tyrosine phosphorylation and actin-enriched lamellipodia on fibronectin were also more prominent than in cells on collagen I. Studies using pharmacological inhibitors or transient transfections revealed that the preferential TGF-β1 effects in cells on fibronectin required c-Src family kinase activity. These observations suggest that a specific cross-talk between TGF-β1 and fibronectin-binding integrin signal pathways leads to the activation of c-Src/Rac1/actin-organization, leading to changes in cell cycle regulator levels in hepatoma cells. Therefore, this study represents another mechanism to regulate cell cycle regulators when integrin signaling is collaborative with TGF-β1 pathways.
Keywords: Integrin; TGF-β1; Cyclin; Signal cross-talk; c-Src; Rac1;

Development of a fluorescent reporter to assess iron regulatory protein activity in living cells by Rebecca J. Henderson; Stephanie M. Patton; James R. Connor (162-168).
Through the insertion of an iron responsive element (IRE) into a pd2ECFP vector, we demonstrate a noninvasive method for determining alterations in iron regulatory protein (IRP) activity that results in changes in protein translation in living cells. This construct takes advantage of the specifically iron-dependent interaction between IRPs that bind IREs on mRNAs to posttranscriptionally regulate protein expression in a manner similar to ferritin production. In this report, we demonstrate, using HEK-293 cells, that an IRE-driven fluorescent reporter can be used to observe changes in cellular iron status that are sufficient to alter protein synthesis. When iron availability was decreased, there was less cyan fluorescent protein (CFP) expression, suggesting that IRPs bind to the IRE and block protein translation. Conversely, exposing the cells to iron increased CFP fluorescence. This construct has advantages over traditionally used dyes and existing IRE driven constructs because it can be used to repeatedly study iron-influenced protein production over extended periods of time. The future applications of this construct include investigation of how mutations in cells may impact cellular iron metabolism and how various types of exogenously applied trophic, stress, and therapeutic agents may impact cellular iron metabolism.
Keywords: Iron regulation; Fluorescent reporter; Ferritin; IRE;

Regulation of HIV-1 env mRNA translation by Rev protein by Celia Perales; Luis Carrasco; Maria Eugenia González (169-175).
We have examined the effect of Rev on the regulation of the expression of RRE containing mRNAs when they were synthesised in the nucleus or directly in the cytoplasm. In the nuclear expression system, Rev enhanced env mRNA transport by about 1.6-fold, while translation of this mRNA was increased more than a 100-fold. These findings indicate that the target of Rev activity is located mainly at the translational level. Synthesis of Env using a recombinant vaccinia virus system, which synthesised env mRNA directly in the cytoplasm, is also enhanced by Rev. Finally, RRE functioning was examined using a luciferase mRNA bearing this element. Rev stimulated the synthesis of Luciferase both when the luc mRNA was made in the nucleus or in cytoplasm. Our results indicate that the effect of Rev on env mRNA transport is low compared with the enhancement of translation of this mRNA.
Keywords: HIV rev; HIV env; Regulation of translation; Rev responsive element;

Differentiation of Drosophila Schneider cells caused by DNA double-strand break (DSB)-inducing topoisomerase II (topo II) inhibitors were attenuated by ICRF-193, a non-DNA-damaging topo II inhibitor. ICRF-193 did not inhibit differentiation induced by neocarzinostatin (NCS), a drug that causes DNA DSBs independent of topo II. Schneider cells differentiated upon treatment with γ-ray. These results suggest that DNA DSBs induce myogenic differentiation of Schneider cells. We also found DNA replication inhibitors, hydroxyurea (HU), aphidicolin, and ethylmethanesulfonate (EMS) induced myogenic differentiation of Schneider cells. HU-induced differentiation was inhibited upon pretreatment of cells with chemical inhibitors of PP 1/2A, p38 MAPK, JNK, and proteasome. RT-PCR analysis revealed that the expressions of fusion-competent myoblast-specific genes lmd, sns, and del were induced in Schneider cells upon treatment with NCS or HU, whereas expressions of three founder cell-specific genes, duf, ants, and rols, were undetectable. These results indicate that the expression of fusion competent-myoblast-specific genes is induced during myogenic differentiation of Drosophila Schneider cells by DNA DSBs or replication inhibition.
Keywords: Drosophila; Myogenesis; Fusion-competent myoblast; Double-strand break; Replication inhibition;