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R R Friis

Publications and source records attributed to R R Friis.

At least 37 records · Page 2Linked to original sources
PubMed1990

Megakaryocytic differentiation of K562 cells is associated with changes in the cytoskeletal organization and the pattern of chromatographically distinct forms of phosphotyrosyl-specific protein phosphatases.

We have analyzed morphological and biochemical changes occurring during megakaryocytic differentiation of the human chronic myelogenous leukemia cell line K562 induced by phorbol 12-myristate 13-acetate (PMA). PMA-treated cells became growth arrested, were slightly larger and irregular in shape, adhered better to the culture flask surface, and expressed the glycoprotein IIIa on their surfaces. The morphological changes induced by PMA treatment were associated with the disappearance of actin from the cytosol and presumably reflect PMA-induced actin polymerization. Megakaryocytic differentiation was accompanied by about a 3-fold decrease in the specific phosphotyrosine protein phosphatase (PTPase) activity in the particulate membrane fraction, whereas the activity in the soluble cytosol fraction increased about 3-fold. The decrease of PTPase activity in the particulate membrane fraction could be attributed to the disappearance of at least 1 distinct PTPase form displaying an apparent native Mr of 200,000 and a reduction in activity of a Mr 43,000 PTPase found associated with membranes of all cells examined to date. The increase of PTPase activity in the cytosol fraction manifested itself by the appearance of a new Mr 40,000 PTPase and a reduction of a Mr 60,000 PTPase. These results suggest the existence of several growth- and/or differentiation-related PTPase activities in K562 cells.

Cell Differentiation↗
PubMed1990

Oxygenation of tumors derived from ras transformed cells.

In order to gain insight into mechanisms governing the development of tumor hypoxia, malignancies derived from spontaneously tumorigenic or ras-transformed cell lines were grown in nude mice. As a rule, tumors with ras oncogenes exhibited rapid growth rates and large areas with low pO2 readings even at small tumor sizes. The slow proliferation rate of a spontaneously tumorigenic cell line was consistent with more adequate tissue oxygen levels. In all lines, hypoxia was accentuated at larger tumor sizes. These results demonstrate that ras transformation can lead to accelerated proliferation rates and is then concomitant with the development of pronounced tumor hypoxia.

Animals↗
PubMed1989

Characterization of a membrane-associated phosphotyrosyl protein phosphatase from the A431 human epidermoid carcinoma cell line.

The A431 human epidermoid carcinoma cell line exhibits a 30-100-fold overexpression of the epidermal growth factor (EGF) receptor. We have characterized a membrane-associated phosphotyrosyl-protein phosphatase (PTPase) in these cells since it seemed reasonable that overexpression of the EGF-receptor tyrosine kinase will be matched by high PTPase activity. Indeed, of 12 cell lines tested, the A431 cells had the highest specific PTPase activity. About 70% of the total cellular PTPase activity was found associated with membranes after cell fractionation. The membrane-associated PTPase was hydrophobic as judged by its behaviour in Triton X-114 phase partitioning. High-performance liquid chromatography (HPLC) on a DEAE column revealed a single, homogeneous species of membrane-associated PTPase with an apparent molecular mass of 43 kDa as determined by HPLC on a gel permeation column in the presence of Triton X-100. Comparison of this PTPase with the membrane-associated PTPase activities present in rat spleen and in the human chronic myelogenous leukemia cell line K562 revealed additional species resolvable by DEAE-HPLC. These findings suggest that cells may possess different PTPase activities depending on their growth and differentiation states.

Animals↗
PubMed1989

New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally.

We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM-2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monoculture, none of the epithelial clones could be induced to synthesize caseins. Coculture of epithelial and fibroblastic clones, however, rendered the epithelial cells competent to differentiate functionally; the addition of lactogenic hormones to these cocultures resulted in the synthesis of beta-casein in amounts comparable to that seen with the original IM-2 line. Using this unique cell system, we have investigated the interrelationships between different steps in differentiation leading to hormone-induced casein production. Independent of hormones, epithelial-fibroblastic cell contacts led to the formation of characteristic structures showing the deposition of laminin. We found that the epithelial cells located in these structures also exhibited significantly increased levels of cytokeratin intermediate filament polypeptides. Double immunofluorescence revealed that the cells inducible by hormones to synthesize casein, colocalized exactly with the areas of laminin deposition and with the cells showing greatly intensified cytokeratin expression. These results suggest that hormone-independent differentiation events take place in response to intercellular epithelial-mesenchymal contacts. These events in turn bring about a state of competence for functional differentiation after lactogenic hormonal stimulation.

Animals↗
PubMed1988

Regulation of type VI collagen synthesis in transformed mesenchymal cells.

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the 'nuclear' oncogene v-myc had an inhibitory effect similar to that of the 'cytoplasmic' oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.

Animals↗
PubMed1988

Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line.

In order to study the hormonal regulation of gene expression in mammary epithelial cells, we isolated a prolactin-responsive cell clone, HC11, from the COMMA-1D mouse mammary epithelial cell line. Clone HC11 was selected as a unique example of a cloned mouse mammary epithelial cell which has no requirement for complex, exogenously added, extracellular matrix or co-cultivation with other cell types for the prolactin-dependent in vitro induction of the endogenous beta-casein gene by lactogenic hormones. Induction of beta-casein mRNA is rapid and was detected 3 h after hormone stimulation. A prolactin-dependent increase in the rate of transcription of the beta-casein gene was shown in an in vitro nuclear transcription assay. beta-Casein protein was detected in an immunoblot assay after 24 h, and further accumulated during 5 days of hormone treatment. To identify low-abundance proteins induced directly after prolactin stimulation, mRNA was accumulated during 5 h of stimulation of HC11 cells with prolactin in the presence of cycloheximide. Following cycloheximide removal, the mRNA was translated into protein during a 60-min [35S]methionine pulse and the proteins were resolved by DEAE ion exchange HPLC and SDS-PAGE. A strong induction of a 120-kd cytosolic protein was detected which was maximally expressed within 6 h of hormone stimulation.

Animals↗
PubMed1988

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent.

We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.

Animals↗
PubMed1984

pp60c-src is a substrate for phosphorylation when cells are stimulated to enter cycle.

The endogenous cellular oncogene products, pp60c-src, exhibits a protein kinase activity, but is itself a phosphoprotein. Based on the assumption that pp60c-src might play a role in the control of cell proliferation, we have studied its behaviour as a substrate for phosphorylation known to occur when quiescent, serum-deprived cells are stimulated to enter cell cycle following addition of either serum, platelet-derived growth factor or the phorbol ester derivative, 12-O-tetradecanoyl-phorbol-13-acetate. For this purpose a partial purification of pp60c-src on DEAE ion-exchange chromatography was combined with immune precipitation. A 2-4-fold increase in serine phosphorylation of pp60c-src was consistently observed after stimulation of quiescent cells to growth.

Animals↗
PubMed1984

Biological and biochemical characterization of a new isolate of feline sarcoma virus: Theilen-Pedersen (TP1-FeSV).

A new feline sarcoma virus designated Theilen-Pedersen (TP1-FeSV) has been isolated from a spontaneous, slowly growing fibrosarcoma of a domestic short-haired 4-year-old castrated cat. The virus codes for a gag-onc fusion protein of 83,000 molecular weight phosphorylated in vivo at serine, threonine, and tyrosine residues. Cells transformed in vitro with TP1-FeSV exhibit five- to 10-fold elevated levels of phosphotyrosine over FeLV-infected cells. The gag-onc polyprotein has associated with it a tyrosyl protein kinase activity which in vitro results in autophosphorylation of the molecule at tyrosine residues. The fusion protein cannot be labeled metabolically with [3H]glucosamine and tunicamycin has no effect on the electrophoretic mobility of the in vivo [32P]orthophosphate-labeled fusion protein. The fusion protein, in common with the gag precursor Pr65gag, can be metabolically labeled with palmitic acid.

Acylation↗
PubMed1984

Rous sarcoma virus p19 and gp35 can be chemically crosslinked to high molecular weight complexes. An insight into virus assembly.

We have used the method of chemical crosslinking in order to determine the spatial interactions between components of Rous sarcoma virus. A high molecular weight complex formed by crosslinking has been isolated by ultracentrifugation on sucrose density gradients containing 0.1% (w/v) sodium dodecyl sulphate. This complex is composed of the two viral glycoproteins gp85 and gp35, the gag protein p19, and the viral RNA. Two types of bonding are important for the formation and stability of the complex: first, native disulphide bonds between gp85 and gp35 and between individual p19 molecules; and second, hetero-crosslinking between gp35 and p19 as well as homo-crosslinking between p19. Although viral RNA is quantitatively present in the complex, experiments with RNase treatment show that it is not essential for its formation or stability. A small amount of lipid is present in the complex and appears to be crosslinked to p19. In vitro-labelling of purified virus with the lipophilic photoactivatable reagent [125I] iodonaphthylazide resulted in the labelling of gp35 and p19/23. In vivo-labelling of virus with [3H]palmitate resulted in only gp35 becoming labelled. These results substantiate the membrane association of these proteins. The significance of the interactions in the high molecular weight complex for the stability of the virus and, by implication, the role which they may play in viral assembly are discussed.

Avian Sarcoma Viruses↗
PubMed1984

Drastic rise of intracellular adenosine(5')tetraphospho(5')adenosine correlates with onset of DNA synthesis in eukaryotic cells.

An assay of adenosine(5')tetraphospho(5')adenosine (Ap4A), based on the luciferin/luciferase method for ATP measurement, was developed, which allows one to determine picomolar amounts of unlabeled Ap4A in cellular extracts. In eukaryotic cells this method yielded levels of Ap4A varying from 0.01 microM to 13 microM depending on the growth, cell cycle, transformation, and differentiation state of cells. After mitogenic stimulation of G1-arrested mouse 3T3 and baby hamster kidney fibroblasts the Ap4A pools gradually increased 1000-fold during progression through the G1 phase reaching maximum Ap4A concentrations of about 10 microM in the S phase. Quiescent 3T3 cells reach a high level of Ap4A (1 microM) in a 'committed' but prereplicative state if exposed to an external mitogenic stimulant (excess of serum) and simultaneously to a synchronizer which inhibits entry into the S phase (hydroxyurea). When the block for DNA replication was removed at varying times after removal of the stimulant decay of commitment to DNA synthesis was found correlated with a shrinkage of the Ap4A pool. Cells lacking a defined G1 phase (V79 lung fibroblasts, Physarum) possess a constitutively high base level of Ap4A (about 0.3 microM) even during mitosis. From this high level, Ap4A concentration increases only about tenfold during the S phase. Temperature-down-shift experiments, using chick embryo cells infected with transformation-defective temperature-sensitive viral mutants(td-ts), have shown that the expression of the transformed state at 35 degrees C is accompanied by a tenfold increase of the cellular Ap4A pool. Treatment of exponentially growing human cells with interferon leads, concomitantly with an inhibition of DNA syntheses, to a tenfold decrease in intracellular Ap4A levels within 20 h. The possibility of Ap4A being a 'second messenger' of cell cycle and proliferation control is discussed in the light of these results and those reported previously demonstrating that Ap4A is a ligand of mammalian DNA polymerase alpha, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.

Adenine Nucleotides↗
PubMed1983

Influence of transformation by Rous sarcoma virus on the amount, phosphorylation and enzyme kinetic properties of enolase.

Using chicken embryo fibroblasts infected with the NY68 transformation-defective temperature-sensitive mutant of Rous sarcoma virus, the phosphorylation and enzyme kinetic properties of enolase have been studied before, and at different stages after, the onset of transformation. A method for purification of enolase was developed, which minimized dephosphorylation. Two enolase (EC 4.2.1.11) isoenzymes were separated by isoelectric focussing revealing that it was the gammagamma form (pI 5.2-6.7) which had become phosphorylated at tyrosine residues after transformation. The phosphorylation of enolase in tyrosine occurred slowly after shift to the permissive temperature, rising from undetectable levels in phenotypically normal cells, to < 10% of the total phosphoamino acid after 3 h, and reaching 30-50% of the total phosphoamino acid by 16 h. Interestingly, the fraction of phosphorylated enolase molecules declined during transformation from 8% in normal cells to 5% by 16 h after temperature shift, due to a 3- to 5-fold increase in the total amount of enolase present in the transformed cultures. Although transformation had no apparent effect on the K0.5 of enolase (26 +/- 4 microM for 2-phosphoglycerate), its specific activity was reduced by about one third.

Animals↗
PubMed1983

Inhibition of Rous sarcoma virus assembly by treatment with 2',5' adenosine nucleotides.

We have investigated the influence of 2',5' adenosine nucleotides on the replication and transformation of cells by Rous sarcoma virus (RSV). Treatment with the nucleotides ppp2',5'A4 and 2',5'A4 causes a striking reduction (50-fold) in the yield of infectious progeny virus, while ppp2',5A2 and 2',5'A3 had virtually no effect. The reduction in infectivity seen with 2',5'A4 nucleotides is paralleled by a smaller but significant (three- to four-fold) reduction in the amount of particles released as measured by reverse transcriptase activity and levels of viral structural proteins. The reduced infectivity of released particles is not due to viral RNA being missing since the amount of genomic RNA in particles from 2',5'A4-treated cultures was likewise only reduced by a factor of 2-3. Pulse-chase radioactive label experiments showed that processing of both viral group-specific antigens (gag) and viral envelope glycoprotein (env) gene products was completely normal in nucleotide-treated cultures, but that the rate of appearance of viral proteins in mature virus in the culture supernatants was reduced by a factor of about 3-4. Taken together, the data show that assembly of viral structural proteins into virions which can be released into the medium is slowed, and that assembly of virus particles with reduced infectivity follows upon nucleotide treatment. This inhibition of infectious virus production takes place without significant toxic effects on the cell; host protein synthesis is only 20% inhibited. There is also no significant effect on the secretory ability of the cells as measured by total protein release into the medium or release of fibronectin. The transformed cell phenotype was also subtly affected by 2',5'A4, but not by other oligomers. Plasminogen activator protease activity was sharply reduced upon treatment, while other typical features of RSV-transformed cells such as elevated hexose transport, and pp60src-associated protein phosphokinase activity, were little affected.

Adenine Nucleotides↗
PubMed1983

Differential expression of endogenous virus glycoprotein in fibroblasts and sera of some adult chickens.

A rapid and convenient assay for the expression of endogenous retrovirus glycoprotein in adult chickens has been developed based on the enzyme-linked immunosorbent assay (ELISA) principle. This method has been standardized using the conventional chick helper factor test. In the course of establishing this method with a large number of specific pathogen-free (VALO) chickens, an interesting diversity became apparent; about 20% of the birds which, according to chick helper factor tests performed with feather follicle fibroblast cultures were negative for endogenous virus glycoprotein expression, exhibited relatively high titres of reactive glycoprotein in serum. However, in no case was a chick helper factor-positive animal negative in serological tests. The possibility of endogenous virus antigen expression which either cannot be detected in fibroblasts, or is incapable of functioning in the chick helper factor complementation, is discussed.

Animals↗
PubMed1983

Structural and kinetic differences between the M2 type pyruvate kinases from lung and various tumors.

The kinetic and structural properties of purified, homogeneous pyruvate kinase type M2 from chicken lung and tumors, including that from Rous sarcoma virus-transformed chicken fibroblasts, have been compared. The "tumor enzyme" is characterized by a low affinity for phosphoenolpyruvate, pronounced serine activation, and strong alanine inhibition as compared to the "lung type". In contrast to the rat lung enzyme, which is not affected by serine, the chicken lung enzyme is slightly activated by serine. The serine metabolites phosphoserine and glycine do not activate the "tumor type M2 pyruvate kinase", but L-alpha-glycerophosphorylcholine and phosphatidylserine do slightly activate at physiological concentrations. Studies with substances structurally related to serine reveal that the hydroxyl group of serine is a prerequisite for the activation and that the amino and carboxyl groups determine the affinity of the "tumor type M2 pyruvate kinase" for serine. Two different fragmentation methods (CNBr-cleavage and V-8 proteolysis) and two different methods for separation of the resulting peptide fragments (polyacrylamide gel isoelectric focussing and SDS-polyacrylamide electrophoresis) indicated a high degree of homology between the type M2 pyruvate kinases from lung and tumors as well the type M1 from muscle. Each type of pyruvate kinase, however, contains one or two unique protein fragments which are characteristic for its type. We have termed those fragments L (lung), M (muscle), and T (tumor).

Animals↗
PubMed1982

The increase in hormone-stimulated adenylate cyclase activity following Rous sarcoma virus transformation.

Rous sarcoma virus (RSV)-infected chicken embryo cells were used to study the effect of viral transformation on the hormone-stimulated synthesis of cyclic AMP. Transformation by RSV greatly increased the cells' ability to synthesize and accumulate cyclic AMP in response to the beta-adrenergic agonist isoproterenol as compared to untransformed cells. This enhancement was observed in both intact cells and in membranes prepared from these cells. The inclusion of guanosine 5'-0-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, in assays of adenylate cyclase activity did not abolish the quantitative differences between the transformed and normal cell membranes. Infection of cells by Rous-associated virus, which lacks the oncogene src, did not induce this hyperresponsiveness thus indicating the probable involvement of the src gene product in this phenomenon. The duration of the isoproterenol-induced cyclic AMP elevation was longer in the transformed than in the untransformed cells; transformed cells, unlike untransformed cells, required at least 120 min before full desensitization became established. Membranes prepared from transformed cells specifically bound more than 5 times the quantity of the beta-adrenergic radiolabeled antagonist (-)3H-dihydroalprenolol and 125I-iodocyanopindolol compared to the untransformed cell membranes. Thus, it appears that major differences between the transformed and normal phenotypes reside in the concentration of membrane beta-adrenergic receptors and the inability of RSV-transformed cells to self-limit their response to specific external stimuli.

Adenylyl Cyclases↗