sodium-dodecyl-sulfate and Cell-Transformation--Neoplastic

Multiple mRNA species encoding several predicted forms of the high-affinity IgE receptor alpha subunit (Fc epsilon RI-alpha) have been previously characterized from rat basophilic leukemia cells. Using the polymerase chain reaction procedure, we have extended these findings to show that one Fc epsilon RI-alpha mRNA variant, characterized by a 163-bp deletion within the coding sequence, exists in normal rat connective tissue mast cells as well as in both transformed and non transformed murine mast cell lines. In addition, a partial murine Fc epsilon RI-alpha genomic clone, spanning the internal-deletion sequence, has been identified, and from analysis of this sequence a mechanism of alternative pre-mRNA splicing is proposed. Finally, mRNA variants have been translated in a cell-free system and the protein products partially characterized.

Topics: Amino Acid Sequence; Animals; Antigens, Differentiation, B-Lymphocyte; Base Sequence; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Genetic Variation; Male; Mast Cells; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Rats, Inbred Strains; Receptors, Fc; Receptors, IgE; RNA, Messenger; Sodium Dodecyl Sulfate; Transcription, Genetic

Transformation-associated protein (TAP) has been detected in MSV-M-transformed rat cell lines as glycosylated, weakly phosphorylated protein of molecular weight (Mr) 66,000 and 68,000. In the ts-MSV-M-transformed rat kidney cell line (6m2), the synthesis of TAP and the v-mos gene product is temperature-sensitive and accompanies the expression of transformation phenotypes. Therefore, TAP potentially plays a role in cellular transformation. On the other hand, SPP represents a family of glycosylated phosphoprotein with apparent Mr ranging from 42,000 to 69,000. SPP has been detected in osteoblasts and in avian and murine retrovirus-transformed rat and mouse epithelial cells. Therefore, the potential relatedness of TAP and SPP was studied. Using the 6m2 cells, we found that SPP was strongly phosphorylated and was synthesized at both the permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. By contrast, TAP was weakly phosphorylated, and was synthesized, as we found previously, only at the permissive temperature of 33 degrees C. Furthermore, in 35S-methionine incorporation studies, TAP became heavily labelled whereas SPP was not (consistent with its amino acid composition having few methionine residues). Using 125I-TAP in both immunoprecipitation and radioimmunoassays, it was found that an antiserum raised against SPP did not cross-react with 125I-TAP. Additionally, SPP has now been found in many human and rodent cells, while TAP thus far has only been detected in MSV-transformed rat cells. These data suggest that structurally, TAP and SPP are not closely related phosphoproteins.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Electrophoresis, Polyacrylamide Gel; Immunosorbents; Iodine Radioisotopes; Kidney; Methionine; Mice; Oncogene Proteins, Viral; Osteopontin; Precipitin Tests; Rabbits; Rats; Sialoglycoproteins; Sodium Dodecyl Sulfate; Sulfur Radioisotopes; Temperature

The secretion of elevated levels of proteinases is considered to be a distinct property of most transformed cells. The cellular and secreted levels of plasminogen activators and collagenases have been examined in the nonmalignant human osteosarcoma (HOS), the malignant Kirsten murine sarcoma virus transformed (KHOS/NP), the temperature sensitive revertant of virus transformed HOS (KHOS-240S) and N-methyl-N'-nitro-N-nitrosoguanidine transformed HOS (MNNG/HOS) clones. Virus and MNNG transformed clones exhibit 100- and 7-fold higher cellular and and 270- and 30-fold higher extracellular plasminogen activator (PA) activity as compared with untransformed HOS controls. The cellular PA activity of the revertant clone is similar to but the secreted level is slightly higher than the HOS controls. SDS-PAGE in the presence of casein and plasminogen is consistent with the major PA species of urinary type (u-PA) and with the absence of PA inhibitor in the parent and revertant clones. The cellular levels of active collagenase are low in all the clones. However, on activation by trypsin, the two active collagenase bands of similar intensity are observed for all the lines in SDS-PAGE in the presence of gelatin. While there appears to be some elevation of secreted collagenase prior to trypsin activation, the activated collagenases appear to have the same size and activity in all of the clones.

Topics: Cell Line; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Humans; Kirsten murine sarcoma virus; Methylnitronitrosoguanidine; Microbial Collagenase; Molecular Weight; Plasminogen Activators; Sodium Dodecyl Sulfate

We analyzed the polypeptide pattern of leukemic cells of infants and older children with acute lymphoblastic leukemia (ALL), using two-dimensional polyacrylamide gel electrophoresis (PAGE). Patterns were analyzed for the occurrence of a previously detected cytosolic polypeptide, designated L3. Quantitative analysis of L3 in 12 infants and 91 older children with non-T ALL indicated lack of expression of polypeptide L3 in leukemic cells of infants which, in most cases, expressed HLA-DR and CD19 and lacked CD10. Quantitative analysis of L3 in relation to cell surface marker expression revealed that L3 was limited in its occurrence to non-T ALL and was not coordinately expressed with any of the surface markers included in the study. Among patients in the HLA-DR-positive, CD19-positive, and CD10-negative group, different levels of polypeptide L3 were observed between infants and older children. These results indicate differences in leukemic cell constituents between infants and older children with ALL and an otherwise similar cell surface marker phenotype.

Topics: Adolescent; Age Factors; Antigens, Surface; Biomarkers, Tumor; Cell Transformation, Neoplastic; Child, Preschool; Electrophoresis, Polyacrylamide Gel; Female; Humans; Infant; Karyotyping; Peptides; Phenotype; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sodium Dodecyl Sulfate

Activation of the oncogenic potential of ras oncogenes occurs by point mutations at codons 12, 13, 59, 61, and 63 of the sequences that codify for its product, a 21-kDa protein designated as p21. This activation has been postulated by computer models as modifiers of the structure of the protein, which may alter its biochemical and biological activities. We have expressed in bacteria the normal ras p21 and five mutated p21 proteins with mutations at positions 12, 59, 61, 12 plus 59, and 12 plus 61. Purification was carried out by solubilization from bacterial pellets in 7 M urea and chromatography through a Sephadex G-100 column to obtain greater than 95% purified proteins. Circular dichroic (CD) spectra showed that the normal protein and that activated by substitution of Ala59 to Thr59 are very similar in their overall structure. By contrast, point mutations affecting either 12 or 61 residues substantially altered the structure of the proteins. When the parameters of Chen et al. [Biochemistry II, 4120-4131 (1972)] were applied to the CD spectra, both normal and thr59-mutated ras proteins showed a less organized structure than mutated proteins at position 12 or 61. Since the Thr59 mutant has more similar transforming activity than other activated proteins, but a GTPase activity similar to that of the normal protein, our results support the hypothesis that there is more than one mechanism of activation of the ras p21 protein. One of these mechanisms involves important structural alterations by point mutations at position 12 or 61 which reduce the GTPase activity of the protein. Another mechanism will be that induced by a substitution of Ala59 to Thr59 which does not substantially alter the protein conformation. A putative alternative mechanism for the activation of this mutant is discussed.

Topics: Cell Transformation, Neoplastic; Circular Dichroism; Dithiothreitol; Membrane Proteins; Mutation; Protein Conformation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Sodium Dodecyl Sulfate

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; G(M1) Ganglioside; Gangliosides; Glycolipids; Glycoproteins; Humans; Mice; Sarcoma Viruses, Murine; Sodium Dodecyl Sulfate

The feeding of carcinogenic 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in the early stages results in a change in the protein composition of the nuclear ribonucleoprotein particles of the rat liver. These particles are associated with newly synthesized RNA and it is assumed that they are involved in the processing and in the transport of this RNA. After 6 weeks of feeding of this azocarcinogen, the amount of one of the main polypeptides (apparent molecular weight 42 000) is decreased and after 10 weeks of feeding the particles are devoid of this polypeptide completely. Feeding of the non-carcinogenic p-aminoazobenzene (AB) is without any effect. The loss of this polypeptide is not characteristic for the malignant transformation. In the nuclear ribonucleoprotein particles isolated from hepatoma which has been induced by 3'-MeDAB this polypeptide is present in even higher proportion to other polypeptides than it is in particles isolated from liver cells of control animals. The 3'-MeDAB binds to the proteins of the liver nuclear ribonucleoprotein particles and interferes with the RNA processing. It is proposed that the changes in the composition of the protein moiety of the particles reflect changes in the population of liver cells leading finally to the selection of hepatoma cells which are resistant to the toxic effect of 3'-MeDAB on RNA processing.

Topics: Animals; Carcinoma, Hepatocellular; Cell Nucleus; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Liver; Liver Neoplasms; Methyldimethylaminoazobenzene; Neoplasm Proteins; Nucleoproteins; p-Dimethylaminoazobenzene; Rats; Ribonucleoproteins; Sodium Dodecyl Sulfate; Spectrum Analysis

Extraction of RNA from animal cells by a method using diethyl-pyrocarbonate yielded 50-60% of the total RNA. RNA purified by a hot phenol-SDS method from adenovirus 2 infected cells showed about 9% homology with adenovirus DNA, and RNA purified by diethyl-pyrocarbonate-SDS showed over 7% hybridization. Profiles of RNA prepared by both methods were identical when studied by polyacrylamide gel electrophoresis.

Topics: Adenoviridae; Animals; Cell Line; Cell Transformation, Neoplastic; Diethyl Pyrocarbonate; Haplorhini; Humans; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Phenols; Rats; RNA; Sodium Dodecyl Sulfate

Topics: Animals; Avian Sarcoma Viruses; Buffers; Cattle; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Dialysis; Electrophoresis; Kidney; Kinetics; Proteins; Serum Albumin, Bovine; Sodium Dodecyl Sulfate; Temperature

Topics: Actins; Animals; Bucladesine; Buffers; Cell Fractionation; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Electrophoresis, Disc; Fibroblasts; Gammaretrovirus; Kidney; L Cells; Membranes; Mice; Mice, Inbred Strains; Microtubules; Myosins; Proteins; Rats; Sodium Dodecyl Sulfate; Subcellular Fractions; Theophylline

Immune precipitation with a monospecific antiserum was employed to study the synthesis of the major viral glycoprotein gp85. Labeled gp85 was detectable by polyacrylamide gel electrophoresis of immune precipitates prepared from lysates of transformed cells which had been labeled for long term with radioactive amino acid or fucose. When immune precipitates were prepared from lysates of cells pulse-labeled with radioactive amino acid, the bulk of the precipitated counts did not appear in gp85 but in a heterogeneous protein fraction with a mean molecular weight of approximately 70,000; this fraction has been designated p70. If, however, the pulse label was followed by incubation of the cells in medium containing excess unlabeled amino acid, the bulk of the precipitated counts comigrated with gp85. Similar pulse-labeling experiments with radioactive fucose and glucosamine suggested that p70 represents incompletely glycosylated precursor to gp85.

Topics: Alpharetrovirus; Amino Acids; Animals; Antigen-Antibody Reactions; Antigens, Viral; Carbon Radioisotopes; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Electrophoresis, Polyacrylamide Gel; Fucose; Glycoproteins; Immune Sera; Immunoassay; Molecular Weight; Precipitin Tests; Rabbits; Sodium Dodecyl Sulfate; Tritium; Viral Proteins

Topics: Animals; Avian Sarcoma Viruses; Cell Fractionation; Cell Transformation, Neoplastic; Chick Embryo; Chromatography, Affinity; Culture Media; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fibrin; Fibrinolysis; Fibroblasts; Hydrogen-Ion Concentration; Iodine Radioisotopes; Isoflurophate; Kinetics; Molecular Weight; Peptide Hydrolases; Plasminogen; Sodium Dodecyl Sulfate; Surface-Active Agents; Tritium

Topics: Animals; Avian Sarcoma Viruses; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Kidney; Molecular Weight; Mutation; Phenotype; Proteins; Rats; Sodium Dodecyl Sulfate; Temperature

The synthesis of two virus-induced proteins, VP1 (mol wt 92,000) and VP2' (mol wt 72,000), was detected in the cell and in purified virions from H-1 parvovirus propagated in synchronous cell cultures.

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Humans; Kidney; Parvoviridae; Simian virus 40; Sodium Dodecyl Sulfate; Viral Proteins; Virus Replication

Topics: Adenine Nucleotides; Animals; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Chemical Precipitation; DNA, Viral; Fibroblasts; Gammaretrovirus; Methods; Micropore Filters; Nucleic Acid Hybridization; Polynucleotides; Rats; RNA-Directed DNA Polymerase; RNA, Viral; Sodium Dodecyl Sulfate; Temperature; Tritium; Uridine

Topics: Animals; Binding Sites; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Cytosol; Dactinomycin; Haplorhini; Kidney; Measles virus; Membranes; RNA, Viral; Sodium Dodecyl Sulfate; Time Factors; Tritium

Several methods have been explored for the detection and characterization of viral proteins from soluble extracts of cells transformed by Rous sarcoma virus (RSV). Viral antigens have been analyzed after gel filtration in several solvents. In addition, immune complexes formed with virus-specific sera have been isolated by agarose gel filtration and by high- or low-speed centrifugation through sucrose solutions. Radioactive proteins from these immune complexes have been analyzed by gel filtration in 6 m guanidine hydrochloride or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Comparison with proteins from purified virus indicates the presence of two viral core proteins (gs1 and gs2) in the soluble fraction from virus-producing chicken cells. In the same fraction from RSV-transformed hamster cells (which do not produce virus), three gs proteins (gs1, gs2, and gs3) could be identified. The soluble viral gs proteins are strongly bound to at least two larger polypeptides in cell extracts. These polypeptides do not appear to be viral in origin and have the property of undergoing a time-dependent aggregation in the extracts. One of these cell-derived proteins, which is present in a variety of uninfected cell types, closely resembles actin.

Topics: Animals; Antigens, Viral; Avian Sarcoma Viruses; Carbon Isotopes; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chromatography, Gel; Complement Fixation Tests; Cricetinae; Electrophoresis, Polyacrylamide Gel; Evaluation Studies as Topic; Fibroblasts; Guanidines; Immunodiffusion; Peptides; Sodium Dodecyl Sulfate; Solvents; Sulfur Isotopes; Tissue Extracts; Tritium; Viral Proteins

A method for preparing large membrane fragments and cell ghosts was developed for uninfected and Rous sarcoma virus-transformed chicken embryo fibroblasts in culture. Membrane proteins were analyzed by electrophoresis in acrylamide gels containing sodium dodecyl sulfate. A major amino-acid-containing component of uninfected cell membranes was greatly diminished in amount or absent in membranes of virus-transformed cells. This component, called MP-1, had an electrophoretic mobility in sodium dodecyl sulfate-containing gels similar to that of a protein of a mol wt of 1.42 x 10(5). MP-1 was not altered by changes in cell growth rate or in cells infected with the nontransforming virus RAV-1.

Topics: Amino Acids; Animals; Avian Sarcoma Viruses; Carbon Radioisotopes; Cell Fractionation; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Centrifugation, Density Gradient; Chick Embryo; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Fucose; Glycoproteins; Molecular Weight; Neoplasm Proteins; Proteins; Satellite Viruses; Sodium Dodecyl Sulfate; Spectrophotometry; Thymidine; Tritium

Topics: Amino Acids; Animals; Carbon Isotopes; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Chromatography, Gel; Electrophoresis; Embryo, Mammalian; Fibroblasts; Glucosamine; Glycopeptides; Glycoproteins; Isotope Labeling; Mice; Neuraminic Acids; Peptide Biosynthesis; Polysaccharides; Pronase; Simian virus 40; Sodium Dodecyl Sulfate; Time Factors; Tritium

Topics: Acetates; Adenosine Triphosphatases; Animals; Carbon Isotopes; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Dihydrolipoamide Dehydrogenase; Electrophoresis; Fibroblasts; Glucosamine; Glycoproteins; Leucine; Mice; Oxidoreductases; Peptides; Polyomavirus; Simian virus 40; Sodium Dodecyl Sulfate; Tritium; Urea

Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Chloroform; Cricetinae; Electrophoresis, Disc; Hydrogen Peroxide; Iodine; Iodine Isotopes; Kidney; L Cells; Lipids; Methanol; Mice; Molecular Weight; Peroxidases; Proteins; Sodium Dodecyl Sulfate

Topics: Animals; Avian Sarcoma Viruses; Butanols; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cricetinae; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Kidney; L Cells; Mercaptoethanol; Mice; Molecular Weight; Peptides; Sodium Dodecyl Sulfate; Spectrophotometry; Trypsin; Urea

Antibodies to disrupted murine sarcoma-leukemia virus (MSV[MLV]) were used to study the synthesis of viral polypeptides in the transformed, virus-producing rat cell line 78A1. When cultures were labeled for 10 min with radioactive amino acids, about 9% of the total labeled proteins were precipitated with antiserum against purified MSV(MLV), and 3 to 4% were precipitated with the same antiserum after it had been absorbed with an extract from uninfected rat cells. The difference is due to the presence in the unabsorbed antiserum of antibodies to cellular proteins that are present in purified virus preparations. Intracellular viral proteins labeled with radioactive amino acids were isolated by immunoprecipitation and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The mobilities of intracellular viral polypeptides were identical to those of the purified virion. However, labeled polypeptides having electrophoretic mobilities lower than that of the major virion polypeptide, the group-specific antigen of molecular weight 31,000, were present in higher proportion in the total cell extract and in the membrane fraction than in the virion. These polypeptides appear to be of cellular origin for they were present only in minute amounts in the immunoprecipitates obtained with the absorbed serum. After a 10-min labeling period, radioactive proteins were assembled into extracellular virions rapidly for the first 4 hr followed by a slower rate. More than 2% of the total proteins of the cell labeled in a 10-min pulse were assembled into virions at the completion of a 24-hr chase. The high-molecular-weight polypeptides with the same mobilities as those detected in the immunoprecipitate of intracellular proteins were found in virions released from cells after a 10-min pulse. A larger proportion of these high-molecular-weight proteins was detected in virions released after short chase periods (30-120 min) than after longer chase periods (6-24 hr). Two possible interpretations of these data are that the high-molecular-weight cell-derived polypeptides (i) have a turnover rate higher than that of the major virion polypeptides or (ii) are cleaved proteolytically from the virions during long incubation in the culture media.

Topics: Amino Acids; Animals; Carbon Isotopes; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cytoplasm; Electrophoresis, Polyacrylamide Gel; Gammaretrovirus; Immunodiffusion; Molecular Weight; Peptide Biosynthesis; Peptides; Rats; Sodium Dodecyl Sulfate; Tritium; Viral Proteins; Virus Replication

Topics: Acrylamides; Burkitt Lymphoma; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; DNA, Neoplasm; Electrophoresis; Genetic Code; Humans; In Vitro Techniques; Lectins; Leukemia; Leukemia, Lymphoid; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Lymphocytes; Neoplasm Proteins; Nucleoproteins; Sodium Dodecyl Sulfate; Thymidine; Tritium

Topics: Adenoviridae; Animals; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Chemical Precipitation; Cricetinae; DNA, Viral; Fibroblasts; Formamides; Haplorhini; Kidney; Nucleic Acid Hybridization; RNA, Viral; Sodium Dodecyl Sulfate; Spectrophotometry; Tritium; Uridine