Growth hormone-dependent changes in the rat lung proteome during alveorization

Growth hormone (GH) mRNA and protein have recently been demonstrated in the rat lung throughout the period of alveolarization (day 4-14 postnatally). The functional significance of this finding was therefore assessed, by determining the effects of GH mRNA knockout using aerosolized antisense oligodeoxynucleotides (ODN) directed against the GH gene. In a preliminary experiment, the effectiveness of the antisense GH ODN was demonstrated in a lung Type II epithelial cell line (L2 cells), in which constitutive GH mRNA expression was completely abolished by GH ODN transfection. Administration of the aerosolized GH ODN to 4-day-old rats for 10 days was accompanied by a widespread presence of its delivery liposomes within lung cells. Aerosolized GH ODN treatment decreased lung concentrations of IGF (insulin-like growth factor)-1 and increased concentrations of albumin, calcyclin binding protein, superoxide dismutase, RNA binding protein motif 3, and the alpha- and beta-subunits of ATP synthase and electron transfer flavoprotein. At least 32 other proteins (identified by 2D gel electrophoresis) were also significantly affected by the antisense GH ODN treatment. By changing the lung proteome, these results indicate hitherto unsuspected autocrine/paracrine actions of GH in developmental lung function.     

Oligonucleotide biological activity: Relationship to the cell cycle and nuclear transport

Previous studies suggest that oligodeoxynucleotide (ODN) cellular uptake is cell cycle-dependent which may have important implications in cancer cell targeting. To further our understanding of ODN transport and activity, this study examines the relationships between the cell cycle, ODN cellular uptake, intracellular transport, and activity. An antisense c-myc ODN 21-mer was used to study ODN cellular uptake in Rauscher erythroleukemia cells synchronized by either chemical methods or flow cytometry. ODN uptake was examined using subcellular fractionation and confocal fluorescence microscopy. Western blot analysis was used to measure ODN-mediated decreases in c-myc protein levels. Intracellular ODN distribution and extent of uptake was influenced by the phase of the cell cycle, but the mechanism of uptake was not. The relative activity of the antisense ODN was positively correlated to ODN distribution to the cytosol, but negatively correlated to total cellular uptake. Although ODN total cellular uptake is positively influenced by the cell cycle, retention of the ODN in the cytosol (presumably extra-vesicularly) appeared to be relevant in determining the activity of an antisense ODN. Novel methods to target cytosol-acting drugs to the cytoplasm may therefore be warrented.     

Blockage of IGF－1R signaling sensitizes urinary bladder cancer cells to mitomycin－mediated cytotoxicity

INTRODUCTIONtransitional cell carcinoma (TCC) of the bladder represents the fifth most preValent malignancy inwestern population. A major problem in the management of TCC is the low sensitivity to chemotherapy and the high recu-rrence after transurethral resection, which occupies a large proportion (approximately 40%) among bladder cancer patients[1, 21. Sodrug resistance remains a major and difficult problem to resolye in TCC chemotherapy. This phenomenon has often been ascribed to so…     

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt''s lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.     

Antisense oligonucleotides reach mRNA targets via the RNA matrix: downregulation of the 5-HT1A receptor

Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on the ease of design, efficiency of (intra)cellular delivery, ODN stability, and target specificity. Equally essential is a detailed understanding of the mechanism of antisense action. To address these issues, we employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, governed by sequence and structure. We demonstrate that rather than various intracellular factors, the gene sequence per se primarily determines the antisense effect, since 5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells are all efficiently downregulated following ODN delivery via a cationic lipid delivery system. The data also reveal that the delivery system as such is a relevant parameter in ODN delivery. Antisense ODNs bound extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than a steric blockage of the mRNA impedes protein expression. The specificity of the antisense approach, as described in this study, is reflected by the effective functional downregulation of the 5-HT1A receptor.     

Antisense inhibition of IGF receptor expression in HaCaT keratinocytes: a model for antisense strategies in keratinocytes

Antisense strategies targeting skin conditions are attractive in concept, with a number of possible pathologic conditions, such a psoriasis, apparently suitable for such an approach. Because in vitro screening of candidate sequences is usually desirable, we have attempted to use a range of new generation cationic lipids to produce significant antisense oligodeoxynucleotide (ODN) uptake in an immortalized keratinocyte cell line (HaCaT). A large number of commercially available lipids were screened for the ability to induce nuclear ODN localization: Tfx-50, Tfx-20, Tfx-10, Superfect, Cytofectin GSV, Perfect lipids 1-8, Lipofectin, and Lipofectamine. All lipids were used at a range of concentrations (1-20 microg/ml) and with a range of ODN concentrations (1-1000) nM). Of all lipids used, only Cytofectin GSV and Superfect produced significant (>30% of cells) levels of nuclear positive cells, with Superfect also producing significant toxicity at the effective concentration used. Only two treatments produced a significant reduction in target mRNA: insulin-like growth factor-1 receptor (IGF-1R)-ODN 64 complexed with Cytofectin GSV (27.1% +/- 3.5% of IGF-1R mRNA in untreated cells,p < 0.01) and ODN 64 complexed with 10 microg/ml Lipofectin (62.2% +/- 3.4% of IGF-1R mRNA in untreated cells, p < 0.05). Only one treatment, ODN 64 complexed with Cytofectin GSV, produced a reduction in cell growth and survival as assessed by amido black assay. These results demonstrate that in HaCaT keratinocytes, Cytofectin GSV alone of all commercially available cationic lipids was effective in delivering antisense ODN into cell nuclei such that a profound antisense effect could be demonstrated.     

Specific binding of insulin-like growth factors 1 and 2 to the type 1 and type 2 receptors respectively.

1. Competitive binding and receptor cross-linking experiments have been used to examine the receptor-ligand interactions between three bovine insulin-like growth factors (IGF) and monolayer cultures of myoblasts and fibroblasts. 2. Labelled IGF-2 bound predominantly to the type 2 receptor with negligible label cross-linked to the type 1 receptor, notwithstanding the ability of IGF-2 to compete effectively for the binding of IGF-1 to the type 1 receptor. Approx. 100-fold higher concentrations of IGF-1 or the N-terminal truncated (des-Gly-Pro-Glu) IGF-1 (-3N:IGF-1) were required to produce competition equivalent to IGF-2. 3. All IGF peptides, but especially IGF-1, enhanced the binding of labelled IGF-2 to the type 2 receptor of lung fibroblasts. This unusual effect was probably a consequence of the displacement of labelled IGF-2 otherwise bound to a medium protein, a conclusion supported by the demonstration of a 38 kDa membrane protein cross-linked to labelled IGF-2. 4. Both IGF-1 and -3N:IGF-1 bound only to the type 1 IGF receptor in L6 myoblasts, rat vascular smooth-muscle cells and human lung fibroblasts. The peptides competed for labelled IGF-1 binding with potencies in the order -3N:IGF-1 greater than IGF-1 greater than IGF-2 much greater than insulin. Since the IGF peptides were equipotent in skin fibroblasts, it was proposed that the apparently higher affinity of -3N:IGF-1 for receptors in the other cell types was instead a consequence of a low affinity of this peptide for the competing 38 kDa binding protein.     

IGF-1 phosphorylates AMPK-alpha subunit in ATM-dependent and LKB1-independent manner

Serine/threonine protein kinase AMP-activated protein kinase (AMPK) is a key metabolic stress-responsive factor that promotes the adaptation of cells to their microenvironment. Elevated concentrations of intracellular AMP, caused by metabolic stress, are known to activate AMPK by phosphorylation of the catalytic subunit. Recently, the tumor suppressor serine/threonine protein kinase LKB1 was identified as an upstream kinases, AMPKKs. In the current study, we found that stimulation with growth factors also caused AMPK-alpha subunit phosphorylation. Interestingly, even an LKB1-nonexpressing cancer cell line, HeLa, exhibited growth factor-stimulated AMPK-alpha subunit phosphorylation, suggesting the presence of an LKB1-independent pathway for AMPK-alpha subunit phosphorylation. In the human pancreatic cancer cell line PANC-1, AMPK-alpha subunit phosphorylation promoted by IGF-1 was suppressed by antisense ataxia telangiectasia mutated (ATM) expression. We found that IGF-1 also induced AMPK-alpha subunit phosphorylation in the human normal fibroblast TIG103 cell line, but failed to do so in a human fibroblast AT2-KY cell line lacking ATM. Immunoprecipitates of ATM collected from IGF-1-stimulated cells also caused the phosphorylation of the AMPK-alpha subunit in vitro. IGF-1-stimulated ATM phosphorylation at both threonine and tyrosine residues, and our results demonstrated that the phosphorylation of tyrosine in the ATM molecule is important for AMPK-alpha subunit phosphorylation during IGF-1 signaling. These results suggest that IGF-1 induces AMPK-alpha subunit phosphorylation via an ATM-dependent and LKB1-independent pathway.     

Specific inhibition of estrogen receptor alpha function by antisense oligodeoxyribonucleotides

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERalpha) on ERalpha protein expression and function. Antisense ERalpha ODN transfected into the ERalpha-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17beta-estradiol (17beta-E2), but not 17alpha-E2, augmented this effect, with a threshold effect at 10(-8) M 17beta-E2. The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERalpha protein. 3H-17beta-E2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17beta-E2. An inhibitory effect on 17beta-E2-dependent cell function was confirmed by the demonstration that ERalpha oligo 7-transfected MCF7-K2 cells failed to exhibit 17beta-E2-stimulated cell proliferation. Exogenous 17beta-E2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERalpha catabolism via the proteosomal pathway, suggesting an effect of 17beta-E2 on the plasma membrane and the existence of different ERalpha degradation pathways in the MCF7-K2 cell subclone. As 17beta-E2 had no effect on ERalpha protein degradation, we conclude that the observed reduction of ERalpha protein levels is due solely to the presence of the antisense ERalpha ODN. Antisense ERalpha ODN molecules, therefore, may form the basis of effective therapies against ERalpha-dependent malignancies.     

The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A(2) (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD(+) co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.     

Disappearance of labelled IGF-2 from plasma of the ovine fetus in late gestation.

The role of IGF-2 in the fetus and its possible influence on fetal growth remains speculative. We investigated the size distribution of unsaturated binding sites for labelled oIGF-2 in ovine fetal plasma. In addition, the disappearance of each form of protein bound IGF-2 in the late gestation ovine fetus (125-135 days, n = 5) was estimated. One minute after injection into the fetal femoral vein, 125IoIGF- circulated in the fetal femoral artery bound primarily to a 50 kDa binding protein. Only a small amount of binding to a 150 kDa binding protein was seen with little to no free IGF-2 present. IGF-2 also circulated in association with a large molecular weight complex (ca. 250 kDa) presumed to be circulating receptor bound IGF-2. The half life of the 250 kDa form of IGF-2 was 385.9 +/- 65.4 min, for the 150 kDa form 308.0 +/- 65.0 min, for the 50 kDa form was 35.5 + 2.6 min and for the free form of IGF-2 was 1.6 +/- 0.6 min. There was no apparent movement of intact IGF-2 out of the fetal circulation into any of the fetal fluids or into the maternal circulation. Similarly there was no consistent placental uptake of IGF-2 from the fetal circulation.     

An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation.

To study the role of a nuclear proto-oncogene in the regulation of cell growth and differentiation, we inhibited HL-60 c-myc expression with a complementary antisense oligomer. This oligomer was stable in culture and entered cells, forming an intracellular duplex. Incubation of cells with the anti-myc oligomer decreased the steady-state levels of c-myc protein by 50 to 80%, whereas a control oligomer did not significantly affect the c-myc protein concentration. Direct inhibition of c-myc expression with the anti-myc oligomer was associated with a decreased cell growth rate and an induction of myeloid differentiation. Related antisense oligomers with 2- to 12-base-pair mismatches with c-myc mRNA did not influence HL-60 cells. Thus, the effects of the antisense oligomer exhibited sequence specificity, and furthermore, these effects could be reversed by hybridization competition with another complementary oligomer. Antisense inhibition of a nuclear proto-oncogene apparently bypasses cell surface events in affecting cell proliferation and differentiation.     

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.     

HIV gp120 binds to mannose receptor on vaginal epithelial cells and induces production of matrix metalloproteinases

Background

During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR) as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs) downstream of HIV gp120 binding to hMR.

Principal Findings

Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line) and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody.

Conclusion

hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the vaginal epithelium.