c-Ha-rasEJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype

Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P₄) were transfected by lipofection with c-Ha-ras^EJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6-week period. Oncogene-transfected cells (ras-LF-1) exhibited differences in morphology and growth pattern relative to vector controls (neo-LF-1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF-1 cells. Transfection of c-Ha-ras^EJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchor-age independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum-deprived SMCs with 5% fetal bovine serum (FBS) increased steady-state levels of c-Ha-ras mRNA in both ras-LF-1 and neo-LF-1 but ras induction was more pronounced in ras-transfected cells. α-smooth muscle (SM) actin gene expression was markedly reduced in ras-transfected cells relative to vector controls. These results show that transfection of c-Ha-ras^EJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor-related signal transduction and tumorigenic potential. © 1994 Wiley-Liss, Inc.     

Concomitant cellular expression of heat shock regulated genes of heapatitis B virus surface antigen and of human growth hormone by a NIH-3T3 cell line

A plasmid carrying a DNA fragment of hepatitis B virus, coding for the pre-S2 and the entire S region of the surface antigen (HBsAg), placed under the control of the promoter of the human 70 kDa heat shock protein gene (hsp70) was introduced into Line 6, a recombinant cell line that was selectedfromNIH-3T3 cellspreviously transfected with a similar construct coding for the human growth hormone cDNA gene (chGH) and with the plasmid pEJ carrying the Ha-ras^EJ activated cellular oncogene. The resulting cell line, EMS8, expressed: (1) hsp70/HBsAg and hsp70/hGH hybrid genes, (2) the human Ha-ras^EJ oncogene, and (3) the neomycin resistance gene, the two last plasmid markers being used for cell selection. EMS8 cells were able to carry outpost-translational modifications of the middle M and the major S envelope proteins of HBV, such as assembly and glycosylation. Accordingly, the cells synthesized and secreted both free and glycosylated M and S viralproteins, and the human growth hormone protein. In addition concomitant expression of HBsAg and hGH proteins as well as their mRNA were detected in EMS8 cells at least up to 72 hr after heat induction instead of 24 hr in the case of hGH in line 6 cells.     

In vitro culture of adult and juvenile bud explants of Passiflora species

Cultivar E23, an F1 hybrid of P. edulis and P. edulis f. flavicarpa is usually propagated by shoot-tip grafting. Various media were tested to evaluate the potential of E23 for in vitro propagation. Adult tissue was difficult to culture and did not respond to media containing low (<10 µM) concentrations of growth regulators. Growth of adult buds on intact stem sections was promoted by 1 week of dark incubation on MS basal medium plus 150 µM 2iP, 200 µM adenine sulphate and 17.1 µM IAA (3 mg l^–1), and further developed into shoots on MS medium plus 4.9 µM 2iP (1 mg l^–1) and 5.7 µM IAA (1 mg l^–1). By contrast, juvenile shoots of E23, and Passiflora species: edulis f. flavicarpa, edulis, alata, caerulea, mollissima, coccinea, herbertiana and suberosa grew rapidly on MS medium plus 10 µM kinetin and 5 µM IAA. Rapid multiplication was achieved on MS plus 20 µM BA, 10 µM kinetin, 5 µM IAA, and roots initiated on MS plus 5 µM IAA.Abbreviations IAA indole-3-acetic acid - 2iP N6-iso pentenyl adenine - BA N6-benzyl adenine     

Inhibition of voltage-dependent Na+ current in cell-fusion hybrids containing activated c-Ha-ras

Summary The electrophysiological properties of EJ (human bladder carcinoma), GM2291 (human fetal lung fibroblast), and of three hybrid cell lines obtained from their cell fusion were investigated using the patch-clamp technique. GM2291 cells, which are nontumorigenic, express voltage-dependent Na⁺ channels. The pharmacology and gating properties of the Na⁺ channels in GM2291 cells are distinct from neuronal and cardiac Na⁺ channels. EJ cells, which are tumorigenic and contain activated c-Ha-ras, express inward rectifier K⁺ channels. The three cell-fusion hybrid lines, named 145 (nontumorigenic), 145L (non-tumorigenic but morphologically altered), and 147TR2 (fully tumorigenic segregant), have been previously shown to express levels of activated c-Ha-ras similar to those of the EJ parental line. Voltage-dependent Na⁺ channels were observed in none of the hybrid cell lines, while inward rectifier K⁺ channels were observed in each of the hybrid cell lines. The possibility that c-Ha-ras inhibits expression of a voltage-dependent Na⁺ channel is discussed.     

Hyperplasia in the rabbit bladder urothelium following partial outlet obstruction. Autoradiographic evidence

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts ofE. coli M15 using a one-step procedure which involved immobilised metal ion chromatography on Ni²⁺-nitriloacetic acid agarose (Ni-NTA). The optimal pH for elution by imidazole was 6.6 and the yield of his-ras protein (greater than 95% pure) was about 4 mg/litreE. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver staining of proteins were employed to search forras-binding proteins present in rat brain cytosol. Chromatography of rat brain cytosol alone on Ni-NTA revealed several protein species which were not readily eluted with imidazole. These are likely to be low-abundance brain metal ion binding proteins. Pre-treatment of rat brain cytosol with Ni-NTA before a second round of chromatography on Ni-NTA removed most of these proteins. Chromatography of a mixture of pre-treated rat brain cytosol and purified his-ras protein revealed four new protein bands with molecular weights of 250, 90, 80 and 70 kDa. These were considered to be candidateras-binding proteins. It is concluded that the use of his-ras and immobilised metal ion chromatography does provide an approach which can be used to identifyras binding proteins present in cellular extracts.Abbreviations his-ras histidine-tagged vHa-ras - Ni-NTA Ni²⁺ nitriloacetic acid agarose - IPTG isopropyl thio--D-galactoside     

In vitro corm production of Gladiolus dalenii and G. tristis

The ability of the summer flowering Gladiolus dalenii Van Geel and the winter flowering G. tristis L. to form corms in vitro was investigated. G. dalenii spontaneously formed corms on a shoot induction medium consisting of the basal medium of Murashige & Skoog (1962) with up to 2.0 mg l^-1 benzyladenine (BA), 3% sucrose and solidified with 2 g l^-1 Gelrite^®. The effect of different BA and sucrose concentrations as well as different temperatures on in vitro corm production of G. tristis was further investigated. The best production of shoots per explant was achieved on a medium containing 0.5 to 1.0 mg l^-1 BA, sucrose concentrations of 6 to 9% and cultured at 15°C. The best corm production was achieved at the same temperature and with the same medium with the exception that BA was omitted from the medium. To test the effect of the osmotic potential on the formation of shoots and corms, sucrose was substituted by mannitol at various concentrations. Sucrose proved to be essential for both shoot and corm production and the use of mannitol had no beneficial effect.     

Phosphorylation of the Protein Encoded by the First Open Reading Frame of the MDG4 Transposable Element (gypsy) by Homologous and Heterologous Casein Kinases Type 2

Homogeneous casein kinase type 2 (CK2) was obtained from oocytes of Rana temporaria and cells of Drosophila melanogaster by chromatography on heparin-Sepharose, phosphocellulose, and Mono Q columns using a Pharmacia FPLC system. The procedure was first successfully used for the purification of CK2 from the Drosophila melanogaster cell culture. It has been shown that the protein encoded by the first open reading frame (ORF) of the gypsy transposable element (MDG4) is an effective protein substrate both for homologous and heterologous CK2 from the oocytes of Rana temporaria in vitro. Both enzymes catalyze the incorporation of two moles of phosphate per mole of protein. The K_m and V_max values for the reaction catalyzed by CK2 from the Drosophila cell culture were 32.5 ± 2.1 nM and 70.97 ± 1.89 nmol/min per µg, respectively, and for CK2 from oocytes, these values were 37.6 ± 2.8 nM and 66.02 ± 2.15 nmol/min per µg, respectively.     

Effect of growth factors and antitumor drugs on normal and Vall2Ha-ras transformed C3H 10T1/2 cell transplasma membrane electron transport and growth

Summary Bothras and the transplasma membrane electron transport system have been implicated in the control of cell growth. Since the loss of growth regulation is at the heart of the cancerous phenotype, we have investigated the effect of factors like antitumor drugs, which inhibit cell growth, and growth factors which either cause or prepare the cells for growth. We have found that growth factors increase the plasma membrane electron transport of the normal cells but not that of the cells which have been transfected with oncogenicras (which have a 2–5-fold faster basal rate). In the presence of adriamycin the activity of the electron transport system is and cell numbers are decreased more in normal cells than in the cells which have been transfected with the oncogenic version of the Harveyras gene. Cisplatin inhibits cell growth but does not inhibit ferricyanide reduction. Ferricyanide alone or in conjunction with EGF or TPA does not stimulate cell proliferation with either cell line in the absence of fetal calf serum, so simple activation of plasma membrane electron transport is not sufficient to activate a complete growth response.Abbreviations EGF epidermal growth factor - TPA phorbol myristate acetate - PDGF platelet derived growth factor - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromides - SDS sodium dodecyl sulfate     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene     

Transformation of mouse mammary epithelial cells with the Ha-ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-α production

An enhanced expression of transforming growth factor-α (TGFα) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGFα production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21^ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression.     

Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees

The activity of a -(1-4)-xylan synthetase, a membrane-bound enzymic system, was measured in particulate enzymic preparations (1,000 g and 1,000–100,000 g pellets) obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatamus) and poplar (Populus robusta). The specific activity (nmol of xylan formed min^–1 mg^–1 of protein) as well as the activity calculated on a per cell basis (nmol of xylan formed min^–1 cell^–1) of this enzymic system, markedly increased as cells differentiate from the vascular cambium to xylem. This increase is closely correlated with the enhanced deposition of xylan occurring during the formation of secondary thickening. The possible control of xylan synthesis during the biogenesis of plant cell wall is discussed.     

Callus production from willow (Salix viminalis L.) protoplasts

Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 10⁶ ± 1.9 · 10⁶ protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 10⁶ protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog (1962) medium - WP medium Woody Plant medium (Lloyd & McCown 1981)     

Nucleus image properties and cell death in MCF-10F cells grown on slide substrates differing in nature and size

Summary The immortalized human breast epithelial cell line MCF-10F is an important tool for studies on experimental tumorigenesis induced by drugs, transfected Ha-ras oncogene, and hormones. Considering that many relevant data have thus far been established only for MCF-10F cells cultivated on glass, and that there are data showing different cell death ratios for tumorigenic cells obtained from benzo[a]pyrene (BP)-transformed MCF-10F cells cultivated on plastic compared with glass, nuclear parameters estimated by image analysis and cell death ratios were compared for cells grown on plastic and glass substrates differing in chamber surface sizes and working culture medium volumes. It was concluded that for slides with a growth size equal to 9.4 cm², plastic substrate was more advantageous than glass for growing MCF-10F cells because although the apoptotic ratios (AR) for the cells grown on plastic are low as it would be expected for nontransformed cells, they are bigger than those reported for the BP-transformed MCF-10F cells cultivated on the same substrate but closer to those of the BP-transformed MCF-10F cells receiving a normal chromosome 17. In addition, the plastic substrate did not induce variable nuclear image results as those found in the latter. The 0.5-cm²-sized chambers on plastic slides proved to be inadequate for cell nuclear image analysis and cell death studies on account of the variable geometric, densitometric, and textural results and ARs produced and the unpublished consideration of a very slow growth rate generated under this growth condition.     

In vitro augmentation of cytotoxicity by N-CWS in peritoneal polymorphonuclear leukocytes (PMNs) induced by PSK,OK-432 or N-CWS

Peritoneal polymorponuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 µg)/head of OK-432 or 200 µg/head ofNocardia rubra cell wall skeleton (N-CWS). Withoutin vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in⁵¹Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 µg/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H₂O₂ production of PSK-induced PMNs was increased by thein vitro addition of 25 µg/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.     

Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT→GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10⁷ normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10¹⁰ normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.     

Production of recombinant endostatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED₅₀) for recombinant endostatin was approximately 0.35 g ml^–1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l^–1 at 6 days of cultivation.     

In vitro cytokinin binding to a particulate cell fraction from protonemata of Funaria hygrometrica

Cytokinin-induced bud formation in moss protonemata is specific for cytokinin bases, their ribosides being relatively inactive. Binding of [³H]benzyladenine (BA) to a 13,000–80,000 x g subcellular fraction from extracts of Funaria hygrometrica (L.) Sibth. was measured by a centrifugation assay. Increasing concentrations of non-radioactive BA decreased the binding proportionally to the logarithm of the BA concentration between 3×10^-8 and 10^-4M. [³H]Zeatin also bound to these fractions, although the extent of binding was not as great as with [³H]BA. Biologically active cytokinins, including BA, zeatin, 6-(3-methyl-2-enylamino)purine (IPA) and kinetin, competed for the binding of [³H]BA, whereas the ribosides of BA, zeatin and IPA competed poorly. Other biologically inactive compounds, such as adenine and 9-methyl-BA, were also ineffective as competitors. The ability to bind BA by the 13,000–80,000 x g fraction was greatly reduced by treatment with 1% Triton X-100, and heat treatment eliminated more than one-half of the binding activity. Competitive binding appeared to be pH-dependent, with maximal activity between pH 6.0 and 6.5. After fractionation by differential centrifugation, the ability to bind cytokinins was not correlated with the RNA content of the fraction and thus probably did not represent binding to ribosomes which has been reported in other plant tissues. Cytokinins also exhibited competitive binding to non-biological materials, e.g., talc. The detailed characteristics of the binding of BA to talc were different from those to the biological fractions. However, the problem remains, in all studies of cytokinin binding, to distinguish between binding that is biologically meaningful, and biological (biologically) non-meaningful physical adsorption.Abbreviations BA N⁶-benzyladenine - IPA 6-(3-methyl-2-enylamino)purine - 9-MeBA N⁶-benzyl-9-methyladenine