Inhibition of tumorigenicity of the teratoma PC cell line by transfection with antisense cDNA for PC cell-derived growth factor (PCDGF, epithelin/granulin precursor)

The PC cell line is a highly tumorigenic, insulin-independent, teratoma-derived cell line isolated from the nontumorigenic, insulin-dependent 1246 cell line. Studies of the PC cell growth properties have led to the purification of an 88-kDa secreted glycoprotein called PC cell-derived growth factor (PCDGF), which has been shown to stimulate the growth of PC cells as well as 3T3 fibroblasts. Sequencing of PCDGF cDNA demonstrated its identity to the precursor of a family of 6-kDa double-cysteine-rich polypeptides called epithelins or granulins (epithelin/granulin precursor). Since PCDGF was isolated from highly tumorigenic cells, its level of expression was examined in PC cells as well as in nontumorigenic and moderately tumorigenic cells from which PC cells were derived. Northern blot and Western blot analyses indicate that the levels of PCDGF mRNA and protein were very low in the nontumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic properties. Experiments were performed to determine whether the autocrine production of PCDGF was involved in the tumorigenicity of PC cells. For this purpose, we examined the in vivo growth properties in syngeneic C3H mice of PC cells where PCDGF expression had been inhibited by transfection of antisense PCDGF cDNA. The results show that inhibition of PCDGF expression resulted in a dramatic inhibition of tumorigenicity of the transfected cells when compared with empty-vector control cells. These data demonstrate the importance in tumor formation of overexpression of the novel growth factor PCDGF.

Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinoderm embryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9+2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [3H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9+2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state.

The proliferative and antiapoptotic effects of substance P are facilitated by formation of a β-arrestin-dependent scaffolding complex

A requirement for scaffolding complexes containing internalized G protein-coupled receptors and β-arrestins in the activation and subcellular localization of extracellular signal-regulated kinases 1 and 2 (ERK1/2) has recently been proposed. However, the composition of these complexes and the importance of this requirement for function of ERK1/2 appear to differ between receptors. Here we report that substance P (SP) activation of neurokinin-1 receptor (NK1R) stimulates the formation of a scaffolding complex comprising internalized receptor, β-arrestin, src, and ERK1/2 (detected by gel filtration, immunoprecipitation, and immunofluorescence). Inhibition of complex formation, by expression of dominant-negative β-arrestin or a truncated NK1R that fails to interact with β-arrestin, inhibits both SP-stimulated endocytosis of the NK1R and activation of ERK1/2, which is required for the proliferative and antiapoptotic effects of SP. Thus, formation of a β-arrestin-containing complex facilitates the proliferative and antiapoptotic effects of SP, and these effects of SP could be diminished in cells expressing truncated NK1R corresponding to a naturally occurring variant.

Inhibition of Endothelial Wound Repair by Dominant Negative Connexin Inhibitors

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-βGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ∼50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.

Proteomic dissection of dome formation in a mammary cell line: Role of tropomyosin-5b and maspin

In this work we extended the study of genes controlling the formation of specific differentiation structures called “domes” formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the β-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC β-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC β-subunit and the rat8 proteins, and is under the negative control of maspin.

Potent Inhibition of Ribulose-Bisphosphate Carboxylase by an Oxidized Impurity in Ribulose-1,5-Bisphosphate1

Oxidation of d-ribulose-1,5-bisphosphate (ribulose-P2) during synthesis and/or storage produces d-glycero-2,3-pentodiulose-1,5-bisphosphate (pentodiulose-P2), a potent slow, tight-binding inhibitor of spinach (Spinacia oleracea L.) ribulose-P2 carboxylase/oxygenase (Rubisco). Differing degrees of contamination with pentodiulose-P2 caused the decline in Rubisco activity seen during Rubisco assay time courses to vary between different preparations of ribulose-P2. With some ribulose-P2 preparations, this compound can be the dominant cause of the decline, far exceeding the significance of the catalytic by-product, d-xylulose-1,5-bisphosphate. Unlike xylulose-1,5-bisphosphate, pentodiulose-P2 did not appear to be a significant by-product of catalysis by wild-type Rubisco at saturating CO2 concentration. It was produced slowly during frozen storage of ribulose-P2, even at low pH, more rapidly in Rubisco assay buffers at room temperature, and particularly rapidly on deliberate oxidation of ribulose-P2 with Cu2+. Its formation was prevented by the exclusion of transition metals and O2. Pentodiulose-P2 was unstable and decayed to a variety of other less-inhibitory compounds, particularly in the presence of some buffers. However, it formed a tight, stable complex with carbamylated spinach Rubisco, which could be isolated by gel filtration, presumably because its structure mimics that of the enediol intermediate of Rubisco catalysis. Rubisco catalyzes the cleavage of pentodiulose-P2 by H2O2, producing P-glycolate.

Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor.

The development of new capillary networks from the normal microvasculature of the host appears to be required for growth of solid tumors. Tumor cells influence this process by producing both inhibitors and positive effectors of angiogenesis. Among the latter, the vascular endothelial growth factor (VEGF) has assumed prime candidacy as a major positive physiological effector. Here, we have directly tested this hypothesis in the brain tumor, glioblastoma multiforme, one of the most highly vascularized human cancers. We introduced an antisense VEGF expression construct into glioblastoma cells and found that (i) VEGF mRNA and protein levels were markedly reduced, (ii) the modified cells did not secrete sufficient factors so as to be chemoattractive for primary human microvascular endothelial cells, (iii) the modified cells were not able to sustain tumor growth in immunodeficient animals, and (iv) the density of in vivo blood vessel formation was reduced in direct relation to the reduction of VEGF secretion and tumor formation. Moreover, revertant cells that recovered the ability to secrete VEGF regained each of these tumorigenic properties. These results suggest that VEGF plays a major angiogenic role in glioblastoma.

Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants.

A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.

Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 beta-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). We have reported that nanomolar concentrations of dexamethasone suppress IL-1 beta-induced iNOS protein expression and production of nitrite, the stable end product of NO formation, without affecting IL-1 beta-triggered increase in iNOS mRNA levels. We now have studied the mechanisms by which dexamethasone suppresses IL-1 beta-stimulated iNOS expression in mesangial cells. Surprisingly, nuclear run-on transcription experiments demonstrate that dexamethasone markedly attenuates IL-1 beta-induced iNOS gene transcription. However, this is counteracted by a prolongation of the half-life of iNOS mRNA from 1 h to 2.5 h by dexamethasone. Moreover, dexamethasone drastically reduces the amount of iNOS protein by reduction of iNOS mRNA translation and increased degradation of iNOS protein. These results indicate that glucocorticoids act at multiple levels to regulate iNOS expression, thus providing important insights into the treatment of inflammatory diseases.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

Inhibition of Hippocampal Regeneration by Adjuvant Dexamethasone in Experimental Infant Rat Pneumococcal Meningitis

Pneumococcal meningitis (PM) causes neurological sequelae in up to half of surviving patients. Neuronal damage associated with poor outcome is largely mediated by the inflammatory host response. Dexamethasone (DXM) is used as an adjuvant therapy in adult PM, but its efficacy in the treatment of pneumococcal meningitis in children is controversially discussed. While DXM has previously been shown to enhance hippocampal apoptosis in experimental PM, its impact on hippocampal cell proliferation is not known. This study investigated the impact of DXM on hippocampal proliferation in infant rat PM. Eleven-day-old nursing Wistar rats (n = 90) were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis. Treatment with DXM or vehicle was started 18 h after infection, concomitantly with antibiotics (ceftriaxone 100 mg/kg of body weight twice a day [b.i.d.]). Clinical parameters were monitored, and the amount of cells with proliferating activity was assessed using in vivo incorporation of bromodeoxyuridine (BrdU) and an in vitro neurosphere culture system at 3 and 4 d postinfection. DXM significantly worsened weight loss and survival. Density of BrdU-positive cells, as an index of cells with proliferating activity, was significantly lower in DXM-treated animals compared to vehicle controls (P < 0.0001). In parallel, DXM reduced neurosphere formation as an index for stem/progenitor cell density compared to vehicle treatment (P = 0.01). Our findings provide clear evidence that DXM exerts an antiproliferative effect on the hippocampus in infant rat PM. We conclude that an impairment of regenerative hippocampal capacity should be taken into account when considering adjuvant DXM in the therapeutic regimen for PM in children.

Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth

Administration of active TG2 to two different in vitro angiogenesis assays resulted in the accumulation of a complex extracellular matrix (ECM) leading to the suppression of endothelial tube formation without causing cell death. Matrix accumulation was accompanied by a decreased rate of ECM turnover, with increased resistance to matrix metalloproteinase-1. Intratumor injection of TG2 into mice bearing CT26 colon carcinoma tumors demonstrated a reduction in tumor growth, and in some cases tumor regression. In TG2 knockout mice, tumor progression was increased and survival rate reduced compared to wild-type mice. In wild-type mice, an increased presence of TG2 was detectable in the host tissue around the tumor. Analysis of CT26 tumors injected with TG2 revealed fibrotic-like tissue containing increased collagen, TG2-mediated crosslink and reduced organized vasculature. TG2-mediated modulation of cell behavior via changes in the ECM may provide a new approach to solid tumor therapy.