Fold Amplification Research Articles

Expression of Urokinase and Its Receptor in Invasive and Non-Invasive Prostate Cancer Cell Lines

We previously reported that extracellular matrix invasion by the prostate cancer cell lines, PC-3 and DU-145 was contingent on endogenous urokinase being bound to a specific cell surface receptor. The present study was undertaken to characterize the expression of both urokinase and its receptor in the non-invasive LNCaP and the invasive PC-3 and DU-145 prostate cells. Northern blotting indicated that the invasive PC-3 cells, which secreted 10 times more urokinase (680 ng/ml per 10(6) cells per 48 h) than DU-145 cells (63 ng/ml per 10(6) cells per 48 h), had the most abundant transcript for the plasminogen activator. This, at least, partly reflected a 3 fold amplification of the urokinase gene in the PC-3 cells. In contrast, urokinase-specific transcript could not be detected in the non-invasive LNCaP cells previously characterized as being negative for urokinase protein. Southern blotting indicated that this was not a consequence of deletion of the urokinase gene. Crosslinking of radiolabelled aminoterminal fragment of urokinase to the cell surface indicated the presence of a 51 kDa receptor in extracts of the invasive PC-3 and DU-145 cells but not in extracts of the non-invasive LNCaP cells. The amount of binding protein correlated well with binding capacities calculated by Scatchard analysis. In contrast, the steady state level of urokinase receptor transcript was a poor predictor of receptor display. PC-3 cells, which were equipped with 25,000 receptors per cell had 2.5 fold more steady state transcript than DU-145 cells which displayed 93,000 binding sites per cell.

High level transient gene expression in human lymphoid cells by SV40 large T antigen boost.

High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.

An Enzymatic Microdetermination Method for Hydroxylase Cofactor

Abstract An enzymatic microdetermination method is described to determine the concentration of biologically active hydroxylase cofactor in crude samples. A small amount of hydroxylase cofactor was oxidized and reduced in a cyclic fashion by phenylalanine 4-monooxygenase (EC 1.14.16.1, PheOHase) and dihydropteridine reductase (EC 1.6.99.7, DPRase) in the presence of excess phenylalanine (Phe) and NADH. The amount of NAD+, accumulated as a result of this cyclic reaction (first cycling), was in proportion to that of the hydroxylase cofactor. The NAD+ was further amplified by an enzymatic amplification reaction, NAD cycling (second cycling) and determined fluorometrically. This double cycling method with more than a 20,000 fold amplification provided extremely high sensitivity, down to 0.02 pmol per assay, and was successfully applied to crude microsamples of pg wet weight.

N-MYC and C-MYC Oncogenes Amplification in Medulloblastomas. Evidence of Particularly Aggressive Behavior of a Tumor with C-MYC Amplification

N-myc and c-myc amplification was investigated in 27 medulloblastomas. DNA was extracted from 19 formalin fixed and paraffin embedded tumors and from fresh frozen tumor tissue in 8 other cases. The results showed no evidence of amplification of N-myc oncogene and only 1 case had a 27 fold amplification of c-myc. Cytogenetically, this neoplasm presented numerous double minute chromosomes (DMs). Moreover, it had an unusual rapidly aggressive course with massive cerebrospinal fluid dissemination unresponsive to intrathecal chemotherapy. Our results indicate a low incidence of N-myc and c-myc gene amplification in medulloblastomas, suggesting that the oncogenic mechanism in these neoplasms is not closely related to DNA gene amplification. C-myc amplification, although not frequently observed, may however provide a growth advantage for medulloblastoma cells in vivo, favoring their rapid dissemination. Medulloblastomas with c-myc activation may represent a subgroup of tumors with a more aggressive behavior.

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells

Activation of protein kinase C (PKC) bu phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat live cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by β-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was loner than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.

Exponential Amplification of Recombinant- RNA Hybridization Probes

We have synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase. Each recombinant consists of a sequence specific for the protozoan parasite, Plasmodium falciparum, embedded within the sequence of MDV-1 RNA, which is a natural template for Qβ replicase. The probe sequence was inserted within a hairpin loop that occurs on the exterior of MDV-1 RNA. The recombinant RNAs hybridize specifically to complementary DNA, despite topological constraints on the probe domain, are replicated at the same rate as MDV-1 RNA, despite their additional length, and are able to serve as templates for the synthesis of a large number of RNA copies. A Qβ replicase reaction initiated with only 0.14 femtograms of recombinant RNA (1,000 molecules) can produce 129 nanograms of recombinant RNA product in 30 minutes. This represents a one-billion fold amplification. Our results demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they would serve the dual role of specific probe and amplifiable reporter.

A comparison of immunocytochemical staining enhancement methods using a rapid microtitre immunocytochemistry assay (MIA)

A method is described which allows rapid and quantitative comparison of immunocytochemical staining procedures. Cells grown and fixed in microtitre plates are probed with increasing dilutions of primary antibody and then stained using the procedures under test; the resulting staining intensities are determined using a microtitre plate reader. The microtitre immunocytochemistry assay (MIA) has been used to compare the sensitivities of enhancement procedures based on immunoperoxidase and immunogold staining. Silver enhancement of DAB staining was found to be the most sensitive technique giving up to 200 fold amplification of the peroxidase staining.

Evaluation of epidermal growth factor receptors in bladder tumours.

Epidermal growth factor (EGF) receptor expression in 31 primary human bladder tumours was quantitated using both structural and functional assays and the EGF receptor gene in the same tumours was analyzed by Southern blot analysis. Immunocytochemical studies using the EGFR1 monoclonal antibody (Mab) showed a significant correlation between EGF receptor levels and the stage and grade of the tumours. Autophosphorylation assays employed to evaluate the receptor's tyrosine kinase activity gave results which in general were consistent with the immunocytochemical data. Using internally controlled immunocytochemical studies with two Mabs and Southern blot analysis of DNA isolated from the tumours, no evidence was obtained for the production of truncated receptors similar to those encoded by the v-erb-B oncogene. Gene amplification was not found in any of the superficial tumours, but one invasive tumour with high EGF receptor expression had an 8-10 fold amplification of the EGF receptor gene. The EGF receptor isolated from this tumour showed a normal pattern of tyrosine phosphorylation at all three major autophosphorylation sites. Our detailed study is consistent with the correlation previously found between EGF receptor expression and stage and grade of bladder tumours, and suggests that at this level of analysis EGF receptors in bladder tumours are not abnormal in structure or size, autophosphorylation activity, or gene structure.

Role of metallothionein in detoxification and tolerance to transition metals.

Animal tolerance to the transition metals cadmium and zinc is hereditary. The evolution to a high level of resistance can be accelerated through mutation and selective pressure. We have studied inbred strains of mice and mutants of Chinese hamster ovary cells resistant to cadmium to further these understandings. Results with whole animals show that the difference in the rate and level of metallothionein accumulation is at most twofold between sensitive and resistant strains. However, with cadmium resistant CHO mutant cells, there is an over 60 fold increase in metallothionein and its mRNA upon induction. These mutants show over 60 fold amplification in metallothionein genes. These results offer a direct contrast in the correlation between elevation of metal resistance and increases in metallothionein between two genetic systems.

Identification of a high-affinity receptor for native human interleukin 1 beta and interleukin 1 alpha on normal human lung fibroblasts.

Native human IL-1 beta and IL-1 alpha stimulated prostaglandin E2 secretion by human embryonic lung fibroblasts at half-maximal concentrations of 3 +/- 1.2 pM (+/- SEM) and 10 +/- 2.3 pM, respectively. In contrast to the 20-50-fold lower affinities previously found for IL-1-R on 3T3 cells as well as murine and human lymphoblastoid lines, monoiodo 125I-IL-1 beta bound to normal human fibroblasts with a Kd of 8.4 +/- 4.1 pM in direct binding experiments, and with a Ki of 11.2 +/- 2.8 pM in competitive binding experiments. IL-1 alpha bound to the receptor identified by 125I-IL-1 beta with a Ki of 50 +/- 18 pM. The receptor exhibited homogeneous affinity for IL-1 beta or IL-1 alpha. The receptor did not recognize IL-2, IFN-gamma, tumor necrosis factor alpha, a functionally related monokine, or bovine acidic fibroblast growth factor, a structurally related mediator. Comparison of the biological response curves and binding curves obtained for IL-1 alpha and IL-1 beta showed that they were parallel and that 10-15% occupancy of the estimated 3,000 sites by either species of IL-1 was sufficient to give half-maximal stimulation of prostaglandin E2 secretion. Thus, the amount of apparent signal amplification observed on fibroblasts was considerably lower than the 100-100,000 fold amplification previously reported for lymphoid lines. Crosslinking experiments revealed a major band with a corrected molecular mass of approximately 80 kD and a minor band of approximately 200 kD. Labeling of these bands was blocked by IL-1 beta and IL-1 alpha but not by IL-2, IFN-gamma, or tumor necrosis factor alpha. These results demonstrate that normal human embryonic lung fibroblasts bear IL-1-R of sufficiently high affinity to mediate their biological responsiveness to low picomolar concentrations of IL-1 beta and IL-1 alpha and are consistent with the existence of a single receptor mediating the biological properties of both human IL-1 species.

A history of cleavage and folding: An example from the Goldenville Formation, Nova Scotia

Temporal relationships between cleavage formation and folding in sedimentary rock sequences can vary from either phenomena preceding the other to both occurring simultaneously. Knowledge of this relationship, however, is required to understand fully the relationship of cleavage orientation to finite strain and the origin of cleavage refraction across competent-incompetent layers. Evidence from the Goldenville Formation provides an example of cleavage formed before folding by ∼50% pressure-solution removal of material, resulting in cleavage planes normal to bedding during active folding. This layer-parallel shortening was accompanied by excess pore pressure that in some cases, exceeded lithostatic pressure, causing formation of bedding-parallel quartz veins with crack-seal textures. Folds developed by a mechanism of differential flexural flow with no loss of coherence across bedding surfaces. En echelon veins formed sequentially across some beds during active folding and subsequently were deformed by various increments of bedding-parallel shear. After folds reached ∼80° limb dips, continued horizontal shortening was accommodated by additional pressure solution, but no recognizable new cleavage was formed. Because cleavage in the Goldenville Formation formed perpendicular to bedding before folding and was passively rotated and extended by bedding-parallel simple shear during fold amplification, the resulting cleavage planes cannot lie in the XY plane of the finite-strain ellipsoid. Differential shear between metagray-wacke and slate beds accounts for the observed cleavage refraction, and the bedding-cleavage angle provides a good estimate of the shearing across these beds during active folding. If cleavage can be shown to postdate folding in other examples, it is possible that cleavage planes will lie in the XY plane of the finite-strain ellipsoid.

Induction of riboflavin-carrier protein in the immature male rat by estrogen: Kinetic and hormonal specificity

The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.

Gene amplification and gene correction in somatic cells.

We used gene transfer to identify frequent genetic rearrangements responsible for activating mutant genes in mammalian cells. We transformed an aprt- tk- cell with a plasmid containing a wild-type aprt gene and a truncated, promoterless tk gene. Transformants that integrate a single copy of this plasmid exhibit the aprt+ phenotype but remain tk-. Tk+ variants result from 20 to 50 fold amplification of the linked plasmid along with significant lengths of flanking DNA. They produce aberrant transcripts such that multiple genes are required to generate sufficient enzyme to convert the cell to the tk+ phenotype. One striking feature of the amplified aprt+ tk+ clones is the frequency (10(-4) ) at which aprt- tk+ mutants appear. These phenotypic requirements are such that the tk gene must remain amplified while all the amplified aprt genes become inactivated. The structure of the amplified DNA indicates that within aprt- cells, all amplified units bear identical mutations. These data suggest that these cells possess an efficient correction mechanism that maintains sequence homogeneity among repeated genetic elements.

Experimentally developed folds in a material with a plannar mineral fabric

The experimental development of folds in a foliated wax/mica aggregate having an anisotropy value (( N 1/ Q 1) ∗ ∗ The coefficients N 1, and Q 1 are, respectively, the maximum and minimum principal values of the compressive stiffness of the material. of between 5 and 10 is compared with a theoretical model of finite fold development in statistically homogeneous anisotropic continua described by Cobbold (1976). A pure shear deformation with the compression parallel to the foliation of the model material resulted in the formation of a train of four folds which became chevron in form. The average foliation orientation during the development of these folds for the most part was in agreement with the theoretical predictions for chevron folds in a material with N 1/ Q 1 between 5 and ∞. Some of the mechanical effects of anisotropy predicted by the theoretical model were clearly demonstrated experimentally such as rapid fold amplification and the development of angular profiles at low and intermediate limb-dips.