DeltaFosB,but not FosB,induces delayed apoptosis independent of cell proliferation in the Rat1a embryo cell line

The fates of Rat1a cells expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand binding domain of human estrogen receptor were examined. The binding of estrogen to the fusion proteins resulted in their nuclear translocation and triggered cell proliferation, and thereafter delayed cell death was observed only in cells expressing ER-DeltaFosB. The proliferation of Rat1a cells, but not cell death triggered by ER-DeltaFosB, was completely abolished by butyrolactone I, an inhibitor of cycline-dependent kinases, and was partly suppressed by antisense oligonucleotides against galectin-1, whose expression is induced after estrogen administration. The cell death was accompanied by the activation of caspase-3 and -9, the fragmentation of the nuclear genome and cytochrome c release from the mitochondria, and was suppressed by zDEVD-fmk and zLEHD-fmk but not zIETD-fmk. The cell death was not suppressed by exogenous His-PTD-Bcl-x(L) at all, suggesting involvement of a Bcl-x(L)-resistant pathway for cytochrome c release.     

Regulated expression of HIV-1 Rev function in mammalian cell lines.

In order to facilitate further investigation of Rev function, we have generated two systems for the inducible expression of Rev in mammalian cell lines (HeLa and U937) using either a tetracycline-regulated promoter or fusion of Rev to a modified form of the hormone binding domain of the estrogen receptor. In the case of the fusion of Rev to the modified hormone binding domain of the estrogen receptor, we demonstrated induction of Rev function in response to tamoxifen administration to levels comparable to that of the unmodified Rev protein. Subsequently, U937 lines were generated that retained the observed pattern of hormone-dependent function of the Rev fusion protein. In the case of the tetracycline-regulated system, cell lines (both HeLa and U937) were generated that displayed tight regulation of Rev. In the case of the HeLa cell lines, they were used for the subsequent generation of stable cell lines expressing either HIV-1 env or chloramphenicol acetyl transferase (CAT) in a Rev-dependent fashion. Using the latter cell lines, we demonstrate the ability to control Rev expression over a broad concentration range and find that, as soon as Rev expression is detectable, induction of Rev-dependent gene expression is also observed.     

Codon-improved Cre recombinase (iCre) expression in the mouse

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.     

CD4 molecules with a diversity of mutations encompassing the CDR3 region efficiently support human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion.

The third complementarity-determining region (CDR3) within domain 1 of the human CD4 molecule has been suggested to play a critical role in membrane fusion mediated by the interaction of CD4 with the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. To analyze in detail the role of CDR3 and adjacent regions in the fusion process, we used cassette mutagenesis to construct a panel of 30 site-directed mutations between residues 79 and 96 of the full-length CD4 molecule. The mutant proteins were transiently expressed by using recombinant vaccinia virus vectors and were analyzed for cell surface expression, recombinant gp120-binding activity, and overall structural integrity as assessed by reactivity with a battery of anti-CD4 monoclonal antibodies. Cells expressing the CD4 mutants were assayed for their ability to form syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. Surprisingly in view of published data from others, most of the mutations had little effect on syncytium-forming activity. Normal fusion was observed in 21 mutants, including substitution of human residues 85 to 95 with the corresponding sequences from either chimpanzee, rhesus, or mouse CD4; a panel of Ser-Arg double insertions after each residue from 86 to 91; and a number of other charge, hydrophobic, and proline substitutions and insertions within this region. The nine mutants that showed impaired fusion all displayed defective gp120 binding and disruption of overall structural integrity. In further contrast with results of other workers, we observed that transformant human cell lines expressing native chimpanzee or rhesus CD4 efficiently formed syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. These data refute the conclusion that certain mutations in the CDR3 region of CD4 abolish cell fusion activity, and they suggest that a wide variety of sequences can be functionally tolerated in this region, including those from highly divergent mammalian species. Syncytium formation mediated by several of the CDR3 mutants was partially or completely resistant to inhibition by the CDR3-directed monoclonal antibody L71, suggesting that the corresponding epitope is not directly involved in the fusion process.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recognition of HLA-Cw4 but not HLA-Cw6 by the NK cell receptor killer cell Ig-like receptor two-domain short tail number 4

NK cells are cytotoxic to virus-infected and tumor cells that have lost surface expression of class I MHC proteins. Target cell expression of class I MHC proteins inhibits NK cytotoxicity through binding to inhibitory NK receptors. In contrast, a similar family of activating NK receptors, characterized by the presence of a charged residue in their transmembrane portion and a truncated cytoplasmic tail, augment lysis by NK cells when ligated by an appropriate class I MHC protein. However, the class I MHC specificity of many of these activating NK receptors is still unknown. Here, we show enhanced lysis of HLA-Cw4 but not HLA-Cw6-expressing cells, by a subset of NK clones. This subset may express killer cell Ig-like receptor two-domain short tail number 4 (KIR2DS4), as suggested by staining with various mAb. It is still possible, however, that these clones may express receptors other than KIR2DS4 that might recognize HLA-Cw4. Binding of KIR2DS4-Ig fusion protein to cells expressing HLA-Cw4 but not to those expressing HLA-Cw6 was also observed. The binding of KIR2DS4-Ig to HLA-Cw4 is weaker than that of killer cell Ig-like receptor two-domain long tail number 1 (KIR2DL1)-Ig fusion protein; however, such weak recognition is capable of inhibiting lysis by an NK transfectant expressing a chimeric molecule of KIR2DS4 fused to the transmembrane and cytoplasmic portion of KIR2DL1. Residue alpha14 is shown to be important in the KIR2DS4 binding to HLA-Cw4. Implications of the role of the activating NK receptors in immunosurveillance are discussed.     

Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of E. coli mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in DeltadacA mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.     

Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion.

As a novel approach to studying the modulation of the polarized epithelial phenotype, we have expressed c-Fos and c-Myc estrogen receptor fusion proteins (c-FosER and c-MycER) in mammary epithelial cells. The hybrid proteins could be activated by estrogen for defined time periods and after the cells had achieved their fully polarized organization. Activation of c-MycER deregulated proliferation but did not affect epithelial polarity. Short-term activation of c-FosER induced the reversible loss of morphological and functional cell polarity. In contrast, long-term stimulation of c-FosER caused the cells to depolarize irreversibly, to invade collagen gels, and to undergo epithelial-fibroblastoid cell conversion. Our data suggest that Fos proteins are important in modulating the epithelial phenotype both in normal tissue development and in invasive processes.     

Identification of unoccupied but transformed nuclear estrogen receptor in cultured mouse Leydig cell

The molecular forms of estrogen receptor (ER) in estrogen-responsive mouse Leydig cell line (B-1) have been examined in relation to their biological activity. ER was predominantly recovered in the nuclear fraction upon homogenization even after cells were precultured in the absence of E2 and Phenol Red. This unoccupied nuclear ER (ERn) whose hormone binding ability was extremely thermostable could be extracted with 0.4 M KCl. This stability enabled us to determine hydrodynamic parameters in the ligand-free condition. The Stokes radius and sedimentation constant of this ERn in high salt condition were 5.5 nm and 6.0S, respectively, resulting in its molecular weight of 140,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ER labeled with [3H]tamoxifen aziridine gave a single band of 65,000 Da, indicating that this ERn had a oligomer structure similar to that of transformed nuclear ER complexed with estrogen in the putative target cells. Therefore, we further examined the possibility that this ERn in B-1 cells can activate estrogen-responsive genes without any aid from estrogen. Estrogen responsive element-thymidine kinase promoter-chloramphenicol acetyltransferase fusion gene (ERE-tk-CAT) was transfected into B-1 cells. CAT activity was enhanced only in cells stimulated with estrogen. It may be concluded from these results that transformed ERn can be formed in the absence of estrogen but that binding to estrogen may be required in order to exert its biological activity.     

Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1.

The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (deltaRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER) (N-BxB-ER cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 4-hydroxytamoxifen. Addition of 4-hydroxytamoxifen to N-BxB-ER cells arrested by density or serum starvation causes reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G2/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G1/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27Kip1 cyclin-dependent kinase inhibitor under all culture conditions tested.     

An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejta et al., Cytometry 29: 286-291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.     

Direct binding of the Na--H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation

The association of actin filaments with the plasma membrane maintains cell shape and adhesion. Here, we show that the plasma membrane ion exchanger NHE1 acts as an anchor for actin filaments to control the integrity of the cortical cytoskeleton. This occurs through a previously unrecognized structural link between NHE1 and the actin binding proteins ezrin, radixin, and moesin (ERM). NHE1 and ERM proteins associate directly and colocalize in lamellipodia. Fibroblasts expressing NHE1 with mutations that disrupt ERM binding, but not ion translocation, have impaired organization of focal adhesions and actin stress fibers, and an irregular cell shape. We propose a structural role for NHE1 in regulating the cortical cytoskeleton that is independent of its function as an ion exchanger.     

Human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation mediated by vascular cell adhesion molecule-1: evidence for involvement of cell adhesion molecules in HTLV-1 biology.

While studying the potential role of vascular cell adhesion molecule-1 (VCAM-1) in infection of endothelial cells by human immunodeficiency virus (HIV), we found that VCAM-1 can mediate human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. Both expression-vector-encoded and endogenously expressed VCAM-1 supported fusion of uninfected cells with HTLV-1-infected cells. Fusion was obtained with cell lines carrying the HTLV-1 genome and expressing viral proteins but not with an HTLV-1-transformed cell line that does not express viral proteins. In clones of VCAM-1-transfected cells, the degree of syncytium formation observed directly reflected the level of VCAM-1 expression. Syncytium formation between HTLV-1-expressing cells and VCAM-1+ cells could be blocked with antiserum against HTLV-1 gp46 and with a monoclonal antibody (MAb) against VCAM-1. Fusion was not blocked by antiserum against HIV or a MAb against VLA-4, the physiological counter-receptor for VCAM-1. The results indicate that VCAM-1 can serve as an accessory molecule or potential coreceptor for HTLV-1-induced cell fusion and provide direct evidence of a role for cell adhesion molecules in the biology of HTLV-1.