phosphorus-radioisotopes and Cell-Transformation--Neoplastic

Polycythemia vera, essential thrombocythemia, and primary myleofibrosis are chronic myeloproliferative neoplasms (MPNs) associated with an increased morbidity and mortality. MPNs are also associated with progression to acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). The "true" rate of transformation is not known mainly due to selection bias in clinical trials and underreporting in population-based studies. The outcome after transformation is dismal. The underlying mechanisms of transformation are incompletely understood and in part remain an area of controversy. There is an intrinsic propensity in MPNs to progress to AML/MDS, the magnitude of which is not fully known, supporting a role for nontreatment-related factors. High doses of alkylating agents, P(32) and combined cytoreductive treatments undoubtedly increase the risk of transformation. The potential leukemogenic role of hydroxyurea has been a matter of debate due to difficulties in performing large prospective randomized trials addressing this issue. The main focus of this review is to elucidate therapy-related leukemic transformation in MPNs with a special focus on the role of hydroxyurea.

Topics: Antineoplastic Agents; Cell Transformation, Neoplastic; Disease Progression; Humans; Hydroxyurea; Janus Kinase 2; Leukemia, Myeloid, Acute; Mutation; Myelodysplastic Syndromes; Phosphorus Radioisotopes; Polycythemia Vera; Primary Myelofibrosis; Survival Analysis; Thrombocythemia, Essential

PCBs are carcinogens, but for many decades it was assumed that PCBs may not possess initiating activity. Initiation is a process that involves changes in the DNA sequence, often, but not exclusively produced through DNA adduction by a reactive compound or reactive oxygen species (ROS). DNA adducts can be detected by (32)P-postlabeling, a method that Dr. Ramesh Gupta co-developed and refined. Today these types of assays together with other mechanistic studies provide convincing evidence that specific PCB congeners can be biotransformed to genotoxic and therefore potentially initiating metabolites. This review will provide an overview of our current knowledge of PCBs' genotoxic potential and mechanism of action, emphasizing the contributions of Dr. Ramesh Gupta during his tenures at the Universities of Kentucky and Louisville.

Topics: Animals; Carcinogens; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; DNA Adducts; DNA Damage; Humans; Isotope Labeling; Liver Neoplasms; Mutation; Phosphorus Radioisotopes; Polychlorinated Biphenyls; Toxicity Tests

The covalent binding of xenobiotics to DNA is an important trigger of the multistage process that leads to carcinogenesis. 32P-postlabeling represents a highly sensitive method for biomonitoring exposure to genotoxic agents and for cancer risk assessment; it is capable of detecting less than one DNA adduct per human genome. Recent improvements to the technique have shown that the resistance of adducted DNA to enzyme digestion may lead to an overestimation of the number of different adducts present in a sample.

Topics: Animals; Base Sequence; Cell Division; Cell Transformation, Neoplastic; DNA Adducts; Genome, Human; Humans; Models, Genetic; Molecular Sequence Data; Mutagenesis; Neoplasms; Phosphorus Radioisotopes; Precancerous Conditions; Radioisotope Dilution Technique; Sensitivity and Specificity; Xenobiotics

Topics: Acute Disease; Adult; Antineoplastic Agents; Bloodletting; Cell Transformation, Neoplastic; Chlorambucil; Female; Humans; Leukemia; Male; Phosphorus Radioisotopes; Polycythemia Vera; Pregnancy; Preleukemia; Prognosis; Pruritus; Uric Acid

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Deoxyribonucleases; Embryo, Mammalian; Haplorhini; Hemolytic Plaque Technique; Hot Temperature; Humans; Kidney; Lung; Mice; Phosphorus Radioisotopes; Ribonucleases; Simian virus 40; Trypsin; Virus Replication

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new

Topics: Animals; Benzo(a)pyrene; Cell Transformation, Neoplastic; Cells, Cultured; Comet Assay; Dihydroxydihydrobenzopyrenes; DNA Adducts; DNA Damage; Embryo, Mammalian; Fibroblasts; Isotope Labeling; Mammals; Mice; Mutagenicity Tests; Phosphorus Radioisotopes

Many genotoxic carcinogens react with the sugar-phosphate backbone in DNA to form phosphotriester (PTE) adducts. These lesions are relatively abundant and persistent for some alkylating carcinogens and may therefore serve as useful biomarkers with which to assess genotoxic exposure and potential mutagenic risk. In the present study, we have developed a 32p postlabeling method that permits analysis of total methyl and/or ethyl PTE in DNA at the femtomole level. The technique is based on the inability of all known nucleolytic enzymes to cleave the internucleotide PTE bond. Consequently, complete digestion of alkylated DNA with these nucleases in the presence of an alkaline phosphatase yields PTE-dinucleoside phosphates. These species are then converted to the corresponding dinucleoside phosphates (dNpdNs) by treatment with alkali to permit subsequent 32p labeling. The resulting labeled dinucleotides (32pd-NpdN) are then analyzed by PAGE. Validation of this method has been carried out using a polydeoxythymidylic acid oligonucleotide containing a site-specific methyl PTE. The method has been applied to the in vitro analysis of calf thymus (CT) DNA treated with dimethylsulfate (DMS) or diethylsulfate (DES) and to the analysis of liver DNA from mice treated in vivo with nitrosodiethylamine. In each case, autoradiograms of the polyacrylamide gels showed the anticipated five bands representing the sixteen labeled dinucleotides, with proportional increases observed as the concentrations of DMS or DES used in the in vitro treatment of CT DNA were increased. The identity and frequency of the nucleosides located 5' to the PTE lesions were obtained by nuclease P1 digestion of the gel-isolated 32pdNpdN species and by analysis of the released labeled mononucleotides, 32pdN, by high-performance liquid chromatography with radioactivity detection. Results obtained from CT DNA treated with DMS or DES showed that the frequency of the four detected nucleotides reflected the normal nucleoside content of CT DNA, indicating the random formation of methyl and ethyl PTE adducts in the in vitro modified DNA. However, studies using liver DNA from three strains of mice treated in vivo with nitrosodiethylamine indicated that the frequency of the thymidine and the 2'-deoxyguanosine 5' to the ethyl PTE was significantly different from the corresponding normal nucleoside content. These results are indicative of (a) the nonrandom formation of ethyl PTE in vivo and/or (b) base sequence-spe

Topics: Alkylating Agents; Animals; Carcinogens; Cell Transformation, Neoplastic; Dinucleoside Phosphates; DNA Adducts; DNA Damage; Isotope Labeling; Mice; Phosphorus Radioisotopes

Topics: Aflatoxin B1; Amino Acid Sequence; Animals; Autoradiography; Carcinogens; Cell Line, Transformed; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Epithelium; Female; Isoelectric Focusing; Liver; Molecular Sequence Data; Neoplasm Proteins; Oncogenes; Phosphoproteins; Phosphorus Radioisotopes; Rats; Rats, Inbred F344; Sequence Homology, Amino Acid; Tropomyosin

The mouse benign keratinocyte cell line 308 was previously shown to have less AP-1 DNA binding and transactivation ability than its malignant variant 10Gy5. Because elevated AP-1 activity in 10Gy5 appears to be critical for its malignant phenotype, we were interested in examining the molecular mechanisms that regulate activator protein 1 (AP-1) in this system. In both 308 and 10Gy5 cells, c-fos, fra-2, c-jun, jun B, and jun D were capable of binding to an AP-1 DNA binding site as determined by antibody clearance gel mobility shift assays. By western analysis, jun B steady-state nuclear and cytoplasmic protein levels were reduced in 10Gy5 cells as compared with 308 cells and jun B steady-state mRNA levels were similar in the two cell lines. The rate of jun B protein synthesis was decreased in 10Gy5 cells in comparison with 308 cells. Gel mobility shift experiments indicated that AP-1 inhibitory proteins were not present in the cytoplasm of 308 cells. Oxidation-reduction posttranslational modification was not a major mechanism of AP-1 regulation in these cells as shown by 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) gel mobility shift assay of nuclear protein treated with a reducing agent and by western analysis for ref-1 protein. Overall phosphorylation of AP-1 proteins in 308 and 10Gy5 cells was examined by 32P orthophosphate labeling and immunoprecipitation. A difference in jun B protein overall phosphorylation was observed in the two cell lines. Our experiments suggest that decreased jun B protein levels may be a mechanism that results in elevated AP-1 activity in malignant 10Gy5 cells.

Topics: Animals; Antibodies; Blotting, Northern; Blotting, Western; Cell Transformation, Neoplastic; DNA; Electrophoresis, Polyacrylamide Gel; Keratinocytes; Methionine; Mice; Mice, Inbred BALB C; Oxidation-Reduction; Phosphorus Radioisotopes; Phosphorylation; Precipitin Tests; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Tumor Cells, Cultured

It has been reported previously that oncogenically transformed cells secrete different molecular forms of osteopontin (OPN), a sialic acid-rich, adhesive, phosphoglycoprotein, than OPNs secreted by their nontransformed counterparts. However, the origin of the OPN isoform secreted by the transformed cells and whether it has different physiological properties which may serve transformation-specific functions remain poorly understood. Here, we report that Rat-1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsB77) secrete two discrete molecular forms of OPN, a 69-kDa OPN at the nonpermissive temperature (41 degrees C) and a 62-kDa form at the permissive temperature (34 degrees C). However, tsB77 cells at both temperatures transcribe a single 1.6 kb OPN mRNA and contain only the 69-kDa form of OPN intracellularly, suggesting that the 69-kDa OPN is modified to the 62-kDa form prior to or immediately after secretion by cells at 34 degrees C. We ruled out proteolytic cleavage, differential phosphorylation, or lack of N- or O-linked carbohydrates as the possible mechanism, but found that the 62-kDa OPN contains significantly reduced levels of sialic acid, as compared to its 69-kDa form. The binding assays using 32P-labeled OPN revealed that only the 69-kDa OPN, not its 62-kDa form, undergoes receptor-mediated localization on the cell surface, although tsB77 cells synthesize OPN receptors (alpha(v)beta3 integrins) at both permissive and nonpermissive temperatures. Furthermore, 125I-labeled purified milk OPN, which is highly sialylated and shows cell surface binding, upon digestion with neuraminidase failed to interact with the cell surface. Taken together, these results suggest that the difference between the 69-kDa and 62-kDa isoforms of OPN resides in their sialic acid content, and sialylation of OPN is crucial for its receptor-mediated binding on tsB77 cells. The data presented here demonstrate for the first time a physiological role of sialic acids in this protein, and raise the possibility that oncogenically transformed tsB77 cells may exploit the lack of OPN-receptor interactions for their invasive behavior.

Topics: Animals; Avian Sarcoma Viruses; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Glycosylation; Humans; Kinetics; Milk Proteins; Molecular Weight; N-Acetylneuraminic Acid; Osteopontin; Peptide Mapping; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Protein Binding; Rats; Receptors, Cell Surface; Serine Endopeptidases; Sialoglycoproteins; Staphylococcus aureus; Temperature; Trypsin; Tumor Cells, Cultured

In polycythemia vera (PV), treatment with chlorambucil and radioactive phosphorus (p32) increases the risk of leukemic transformation from 1% to 13-14%. This risk has been estimated to be 1-5.9% with hydroxyurea (HU) therapy. When compared with historical controls, the risk with use of HU does not appear to be statistically significant. The leukemogenic risk of HU therapy in essential thrombocytosis (ET) and in myelofibrosis with myeloid metaplasia (MMM) is unknown. HU remains the main myelotoxic agent in the treatment of PV, ET, and MMM. We studied 64 patients with these three disorders, seen at our institution during 1993-1995. The patients were studied for their clinical characteristics at diagnosis, therapies received, and development of myelodysplasia or acute leukemia (MDS/AL). Forty-two had PV, 15 ET, and 6 MMM, and 1 had an unclassified myeloproliferative disorder. Of the 42 patients with PV, 18 were treated with phlebotomy alone, 16 with HU alone, 2 with p32, 2 with multiple myelotoxic agents, and 2 with interferon-alpha (IFN-alpha). Two patients from the phlebotomy-treated group, one from the HU-treated group, and 1 from the multiple myelotoxic agent-treated group developed MDS/AL. In the larger group, 11 received no treatment or aspirin alone, 18 were treated with phlebotomy alone, 25 with HU, 5 with multiple myelotoxic agents, 2 with p32, 2 with IFN-alpha, and 1 with melphalan. Study of the entire group of 64 patients showed that only one additional patient (total of 5 out of 64) developed MDS/AL. This patient had been treated with HU alone. Statistical analysis did not show any association between clinical characteristics at diagnosis, or HU therapy, and development of MDS/AL (P=0.5). Thus, our data provide no evidence suggestive of increased risk of transformation to MDS/AL with HU therapy in PV, ET, and MMM. Larger, prospective studies are needed to study this issue further.

Topics: Acute Disease; Anemia, Refractory, with Excess of Blasts; Busulfan; Cell Transformation, Neoplastic; Chlorambucil; Cohort Studies; Disease Progression; Drug Therapy, Combination; Enzyme Inhibitors; Female; Humans; Hydroxyurea; Incidence; Interferon-alpha; Leukemia; Leukemia, Radiation-Induced; Male; Melphalan; Middle Aged; Phlebotomy; Phosphorus Radioisotopes; Polycythemia Vera; Preleukemia; Primary Myelofibrosis; Retrospective Studies; Ribonucleotide Reductases; Risk; Thrombocythemia, Essential

The cyclodiene pesticide chlordane has been reported to be a non-genotoxic carcinogen in rodents. The effects of chlordane on SHE cell transformation were investigated in this study. It appeared that chlordane exhibited a weak transforming activity when applied repeatedly at 8 micrograms/ml. No effect resulted from the combination of benzo[a]pyrene-chlordane. In contrast, chlordane in the range 5-20 micrograms/ml and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1 micrograms/ml) highly potentiated each other when applied sequentially. The synergistic effects could be inhibited by dexamethasone. These results led us to study the genotoxicity of chlordane on SHE cells: no DNA adduct formation could be detected in SHE cells treated with chlordane at a concentration potentiating the transforming effect of TPA. We also confirmed that this pesticide markedly inhibited intercellular communication between SHE as well as V79 cells. These results support literature data on the non-genotoxicity of chlordane. Overall, this study highlights the fact that interaction between-non genotoxic carcinogens may enhance the transformation frequency of SHE cells. Thus, combined effects must be taken into account in the evaluation of carcinogenic risk.

Topics: Animals; Autoradiography; Carcinogens; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Chlordan; Cricetinae; DNA Adducts; Drug Synergism; Embryo, Mammalian; Mesocricetus; Phosphorus Radioisotopes; Tetradecanoylphorbol Acetate

The major routes of metabolic activation of dibenz[a,h]-anthracene (DBA) have been studied in transformable C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts in culture. The morphological transforming activities of three potential intermediates formed by metabolism of DBA by C3H10T1/2 cells, trans-3,4-dihydroxy-3,4-dihydro-DBA-(DBA-3,4-diol), trans-dihydroxy-3,4-dihydro-DBA-anti-1,2-oxide (DBA-3,4-diol-1,2-oxide) and DBA-5,6-oxide were determined. DBA-3,4-diol-1,2-oxide was a strong morphological transforming agent giving a mean of 73% dishes with Type II or III foci and 1.63 Type II and III foci per dish at 0.5 microgram/ml. DBA-3,4-diol produced a mean of 42% dishes with Type II or III foci and 0.81 Type II and III foci per dish at 2.5 micrograms/ml. DBA gave a mean of 24% dishes with Type II or III foci and 0.29 Type II and III foci per dish at 2.5 micrograms/ml. DBA-5,6-oxide was found to be inactive. DNA adducts of DBA, DBA-3,4-diol, DBA-3,4-diol-1,2-oxide, DBA-1,4/2,3-tetrol and DBA-5,6-oxide in C3H10T1/2 cells were analyzed by 32P-postlabeling method. DBA gave 11 adducts, nine of which were observed in the DNA of cells treated with DBA-3,4-diol and seven from cells treated with DBA-3,4-diol-1,2-oxide. Two of these adducts that appear in each of the treatment groups have been identified as the product of the interaction of DBA-3,4-diol-1,2-oxide with 2'-deoxyguanosine. Furthermore, there is evidence for DBA-DNA adducts in cells treated with DBA, DBA-3,4-diol and DBA-3,4-diol-1,2-oxide arising from metabolism to (+,-)-trans,trans-3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-DBA (DBA-3,4,10,11-bis-diol). These results are based on co-migration of C3H10T1/2 DNA adducts with skin DNA adducts formed after topical treatment of mice with DBA-3,4,10,11-bis-diol. In C3H10T1/2 cells, DBA is metabolically activated through DBA-3,4-diol, which is further activated via the DBA-3,4-diol-1,2-oxide and DBA-3,4,10,11-bis-diol pathways. No evidence is provided for the metabolism of DBA by the K-region pathway.

Topics: Animals; Benz(a)Anthracenes; Biotransformation; Cell Transformation, Neoplastic; DNA Adducts; Fibroblasts; Isotope Labeling; Mice; Mice, Inbred C3H; Oxidation-Reduction; Phosphorus Radioisotopes; Structure-Activity Relationship

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T.

Topics: Actins; Animals; Antigens, Polyomavirus Transforming; Cell Line; Cell Transformation, Neoplastic; Fibroblasts; Genetic Vectors; Histocytochemistry; Isotope Labeling; Morphogenesis; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Rats; Recombinant Proteins; Simian virus 40; Transfection

We previously reported that H-ras-induced metastatic ability in murine NIH 3T3 cells is accompanied by increased expression of osteopontin (OPN). OPN is a secreted phosphoprotein that contains a GRGDS amino acid sequence, suggesting adhesive function, but the function of OPN in tumor cells remains poorly understood. Here we report that PAP2 cells (ras-transformed, metastatic NIH 3T3 cells) adhere and spread on OPN-coated substrates, while NIH 3T3 cells adhere and spread poorly on OPN. A similar pattern was seen for adhesion to laminin, while both cell lines adhered equally well to fibronectin. Adhesive interactions to OPN, laminin, and fibronectin were specific and were blocked by GRGDS (but not control GRGESP) peptides. The kinetics of adhesion to all three substrates was examined. Maximum adhesion was observed at 30-60 min, with reduced adhesion thereafter. We also purified metabolically labeled [32P]OPN secreted by PAP2 cells. Labeled OPN bound better in solution to PAP2 cells than to NIH 3T3 cells, and binding to both cell lines was blocked by GRGDS peptides, results that are consistent with the adhesion and spreading of these cells to OPN-coated substrates. Malignant PAP2 cells thus not only secrete increased levels of OPN, relative to NIH 3T3 cells, but also adhere better to this protein. While the target of OPN secreted by tumor cells is not known, our results raise the possibility that tumor cells that secrete OPN may also bind this protein and that this binding may function in autocrine-type signal transduction important to malignancy.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Binding Sites; Cell Adhesion; Cell Adhesion Molecules; Cell Transformation, Neoplastic; Fibronectins; Genes, ras; Laminin; Mice; Molecular Sequence Data; Neoplasm Metastasis; Oligopeptides; Osteopontin; Phosphorus Radioisotopes; Protein Binding; Sensitivity and Specificity; Sialoglycoproteins

The effect of platelet-derived growth factor (PDGF) on c-fos mRNA transcription was studied in the immortalized mouse embryo fibroblast C3H/10T1/2 Cl 8 (10T1/2) cells and the chemically transformed, tumorigenic subclone C3H/10T1/2 Cl 16 (Cl 16). In the 10T1/2 cells as well as the Cl 16 subclone, the dose-dependent PDGF stimulation of c-fos mRNA synthesis was similar in both logarithmically growing and confluent cultures. c-fos mRNA was induced severalfold by 12-O-tetradecanoylphorbol-13-acetate (TPA) in both 10T1/2 and Cl 16. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment inhibited PDGF-stimulated c-fos mRNA expression in Cl 16 cells but did not affect this induction in the 10T1/2 cells. This inhibition was not a general phenomenon of 3-methylcholanthrene-mediated transformation of 10T1/2 cells since experiments with another transformed 10T1/2 cell clone, C3H/10T1/2 TPA 482, gave qualitatively the same results as the 10T1/2 cells. Receptor binding experiments showed that the nontransformed and transformed cells had a comparable number of PDGF receptors, 1.3 x 10(5) and 0.7 x 10(5) receptors per cell, respectively. Furthermore, cAMP-induced c-fos expression induced by forskolin is formerly shown to be independent of PKC down-regulation. In our experiments, forskolin induced c-fos expression in both clones. However, PKC down-regulation inhibited the forskolin-induced c-fos expression in Cl 16 cells. This apparently demonstrates cross talk between PKC and PKA in the c-fos induction pathway. The present results provide evidence for an impaired mechanism for activating c-fos expression through PKC-independent, PDGF-induced signal transduction in the chemically transformed Cl 16 fibroblasts compared to that in nontransformed 10T1/2 cells.

Topics: Adenosine Triphosphate; Animals; Blotting, Northern; Cell Transformation, Neoplastic; Clone Cells; Deoxycytosine Nucleotides; DNA Probes; DNA Replication; Genes, fos; Kinetics; Mice; Mice, Inbred C3H; Phosphorus Radioisotopes; Platelet-Derived Growth Factor; Protein Kinase C; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; RNA; RNA, Messenger; Swine; Tetradecanoylphorbol Acetate; Thymidine

The GTP binding (G) proteins of normal (FRTL5) and ras-transformed thyroid cells (KiKi) were characterized by cholera and pertussis toxin-induced ADP-ribosylation and immunoblot analysis. Two pertussis toxin substrates with molecular masses of 40 and 41 kDa were identified in normal cells as the alpha i2 and alpha i3 subunits. The molecular masses of the cholera toxin substrates were 42 and 45 kDa. The same cholera and pertussis toxin substrates were present in the K-ras-transformed cell line. However, the toxin-dependent ADP-ribosylation was markedly higher in KiKi than in normal cell membranes (more than 50-fold). The reason for this difference was investigated; it could not be explained by the relative amounts of G proteins in the two cell systems, since the levels of alpha i2 subunit as measured by quantitative immunoblot in K-ras-transformed cells were only slightly (65%) higher than in normal cells. The difference in ADP-ribosylation was not due to poly-ADP-ribosylation nor to a different degree of subunit dissociation of G proteins in the two cell lines. Rather, the enhanced ADP-ribosylation in K-ras-transformed cells appears to be due to the loss of an inhibitory factor present in the normal cells. Partial characterization indicates that such a factor is a peripheral membrane protein of less than 25 kDa capable of directly interfering with the ADP-ribosylation reaction.

Topics: Adenosine Diphosphate Ribose; Animals; Brain; Cattle; Cell Line, Transformed; Cell Membrane; Cell Transformation, Neoplastic; Cholera Toxin; Genes, ras; GTP-Binding Proteins; Humans; Kinetics; Macromolecular Substances; Membrane Proteins; NAD; Pertussis Toxin; Phosphorus Radioisotopes; Rats; Thyroid Gland; Virulence Factors, Bordetella

Topics: Animals; Antigen-Antibody Complex; Autoradiography; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chromatography, Affinity; Fibroblasts; Glycoproteins; Immunoblotting; Methionine; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphotyrosine; Radioisotope Dilution Technique; Sulfur Radioisotopes; Tyrosine

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Nuclear Proteins; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Subcellular Fractions; Sulfur Radioisotopes; Tyrosine

C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.

Topics: Aflatoxin B1; Aflatoxins; Animals; Aryl Hydrocarbon Hydroxylases; Benzo(a)pyrene; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Clone Cells; Cytochrome P-450 Enzyme System; DNA; DNA Adducts; Enzyme Induction; Fibroblasts; Male; Mice; Phosphorus Radioisotopes; Rats; Rats, Inbred Strains

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Enzyme Activation; Fibroblasts; Genes, ras; Kinetics; Phosphates; Phosphatidylcholines; Phosphatidylinositols; Phosphorus Radioisotopes; Protein Kinase C; Rats; Transfection

This study deals with the mechanism of the inhibitory effect exerted by the immunosuppressant ciclosporin (CsA) on phorbol-ester-induced inflammation, epidermal hyperplasia and tumor promotion in mouse skin in vivo. This effect coincides with an inhibition of the phosphorylation of a 100-kilodalton protein (p100) in epidermal cytosol in vitro, which has been identified as elongation factor 2 (EF-2) of protein biosynthesis. Phosphorylation of EF-2 is dependent on Ca2+ and calmodulin, and inhibition of EF-2 phosphorylation by CsA is due to an interaction of CsA with calmodulin. The EF-2 phosphorylation system has a metabolic half-life of 1.5 h probably due to a rather rapid turnover rate of the EF-2 kinase. Since CsA inhibits specifically 12-O-tetradecanoylphorbol-13-acetate (TAP)-stimulated but not basal protein synthesis in epidermis, it is proposed that Ca2+/calmodulin-dependent phosphorylation of EF-2 is involved in the induction of the hyperplastic response by TPA and that CsA suppresses TPA effects by inhibition of EF-2-phosphorylation and perhaps other calmodulin-dependent processes. The potential applicability of calmodulin inhibitors in the treatment of hyperproliferative skin diseases is discussed.

Topics: Animals; Calcium; Calmodulin; Cell Transformation, Neoplastic; Chromatography, DEAE-Cellulose; Cocarcinogenesis; Cycloheximide; Cyclosporins; Cytosol; Electrophoresis, Polyacrylamide Gel; Mice; Peptide Elongation Factor 2; Peptide Elongation Factors; Phosphorus Radioisotopes; Phosphorylation; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.

Topics: Amino Acids; Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Chick Embryo; Cytoskeletal Proteins; Fibroblasts; Mutation; Peptide Mapping; Phosphorus Radioisotopes; Phosphorylation; Talin; Trypsin; Tyrosine

The effect of malignant transformation of cells on phosphatidylinositol metabolism was investigated using C3H10T1/2 cells and its chemically transformed cell line, MCA CL-16 cells. We found that incorporation of [32P]Pi into polyphosphoinositide was greatly increased in the transformed cells. A similar tendency was observed when myo-[2-3H]inositol was used as a labelling reagent. It is also observed that influx of labelled inorganic phosphate is enhanced 2-fold by the cell transformation. Therefore, promotion of polyphosphoinositide labelling in the transformed cell might be caused not only by the enhanced metabolism of phosphatidylinositol but also by the increased membrane permeability for radioactive labelling reagents.

Topics: Animals; Biological Transport; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Inositol; Kinetics; Mice; Mice, Inbred C3H; Phosphates; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorus Radioisotopes; Tritium

Using affinity chromatography with immobilized gelatin and native type I collagen, we have identified the major collagen-binding proteins in Nonidet P-40 extracts of chick embryo fibroblasts labeled with [35S] methionine. After washing the gelatin- or collagen-Sepharose beads with high ionic strength buffer, a 47,000-dalton protein was the only major protein besides fibronectin found to bind to these affinity beads. The isoelectric point of this protein was approximately 9.0, with a closely spaced minor spot. The total amount and the synthesis of this collagen-binding protein were both decreased in Rous sarcoma virus-transformed cells. This collagen-binding protein was found to be phosphorylated by incubating intact cells with [32P]orthophosphate. Phosphoamino acid analysis revealed that serine and threonine residues were phosphorylated, but tyrosine was not. Although quantities of the 47,000-dalton protein labeled with [35S]methionine were decreased by a factor 2.5 after transformation, the incorporation of [32P]orthophosphate/unit of protein was 5-7-fold higher in transformed cells. In temperature-sensitive mutant virus-infected cells, the amount of the 47,000-dalton protein was also decreased at the temperature permissive for transformation, and the incorporation of [32P]orthophosphate/protein was also increased. These studies establish that a major membrane-associated collagen-binding protein of fibroblasts is phosphorylated and that it is altered in both total quantity and degree of phosphorylation after malignant transformation.

Topics: Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chromatography, Affinity; Fibroblasts; Molecular Weight; Mutation; Phosphorus Radioisotopes; Phosphorylation; Receptors, Collagen; Receptors, Immunologic; Sulfur Radioisotopes; Temperature

The transforming protein of the Abelson murine leukaemia virus encodes a protein-tyrosine kinase. Previously, we have shown that in Abelson-transformed cells, the Abelson kinase regulates the phosphoserine content of ribosomal protein S6. Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, induces the phosphorylation of S6 at the same five phosphopeptides as found in S6 isolated from Abelson-transformed cells. We have investigated three models whereby the Abelson kinase might regulate S6 phosphorylation via the activation of protein kinase C. First, the Abelson kinase could phosphorylate protein kinase C on tyrosine. However, we do not detect significant amounts of phosphotyrosine in protein kinase C in vivo. Second, it has been suggested that protein-tyrosine kinases might phosphorylate phosphatidylinositol. This could increase the intracellular levels of diacylglycerol and thereby activate protein kinase C. Our data strongly suggest that direct phosphorylation of phosphatidylinositol by the Abelson protein-tyrosine kinase has no physiological role. Third, an indirect activation of protein kinase C may occur via an increase in the rate of phosphoinositide breakdown. We have found that phosphoinositide breakdown appears to be constitutively activated in Abelson-transformed cells. The implications of these observations are discussed with regard to S6 phosphorylation and the mechanism of Abelson-induced transformation.

Topics: Abelson murine leukemia virus; Adenosine Triphosphate; Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Genes; Genes, Viral; Kinetics; Leukemia Virus, Murine; Mice; Mice, Inbred Strains; Phosphatidylinositols; Phosphorus Radioisotopes; Phosphorylation; Protein-Tyrosine Kinases

We have examined the large T encoded by an SV40 mutant, d10, which fails to localize to the nucleus. The DNA sequence of the mutant predicts the alteration of Lys 128----Thr within the sequence 127 Lys Lys Lys Arg Lys 131 of large T. The results show that d10 large T is capable of binding to SV40 DNA, to cellular DNA and to the cellular phosphoprotein p53 as well as wild-type large T. These data suggest that the cytoplasmic location of d10 large T is not due to an inability of the protein to be retained within the nucleus, but argues instead that the protein fails to reach the nucleus because it contains a defective nuclear location signal.

Topics: Adenosine Triphosphate; Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Cell Line; Cell Transformation, Neoplastic; DNA Restriction Enzymes; Mice; Neoplasm Proteins; Nucleoproteins; Oncogene Proteins, Viral; Phosphoproteins; Phosphorus Radioisotopes; Plasmids; Protein Binding; Protein Kinases; Simian virus 40; Tumor Suppressor Protein p53

To facilitate the identification of phosphotyrosine (P-tyr)-containing proteins, rabbit polyclonal antibodies and mouse monoclonal antibody specifically reactive to P-tyr were prepared by hyperimmunizing the animals with P-tyr-conjugated bovine serum albumin or poly-L-lysine. As determined by a solid-phase radioimmunoassay and an enzyme-linked immunosorbent assay, the antibodies reacted with P-tyr-conjugated target antigens but not with those conjugated with phosphoserine (P-ser) or phosphothreonine (P-thr). This immune reaction was strongly blocked by 2 mM P-tyr and phenylphosphate but not by P-ser or P-thr. The antibodies were capable of isolating, as the major P-tyr-containing components, a 170kd protein (most likely the EGF receptor) from EGF-stimulated, 32P-labelled A431 cells, and 130kd and 60kd proteins from Rous sarcoma virus (RSV)-transformed chick cell lysate which had been labelled in vitro with gamma-32P-ATP. Immunofluorescent staining of RSV-transformed cells and A431 cells showed specific localization of P-tyr-containing proteins in the cytoplasm, plasma membrane, and nucleolus-like structures. The results demonstrated the usefulness of the antibodies for identification or isolation of P-tyr-containing proteins.

Topics: Animals; Antibodies; Antibody Specificity; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Chick Embryo; ErbB Receptors; Female; Fluorescent Antibody Technique; Mice; Phosphorus Radioisotopes; Phosphotyrosine; Proteins; Rabbits; Receptors, Cell Surface; Tyrosine

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

Topics: Animals; Avian Sarcoma Viruses; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Fibroblasts; Kinetics; Molecular Weight; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Protein Tyrosine Phosphatases

A 32P-postlabeling method for carcinogen-DNA adduct analysis recently developed in our laboratory was applied to skin DNA from mice treated topically with polycyclic aromatic hydrocarbons (PAHs). After application of 4 doses of 1.2 mumol each of benzo[alpha]pyrene (BP), 3-methylcholanthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA), respectively, total covalent adduct binding in mouse skin DNA initially amounted to 1 adduct in 6.0 X 10(4) - 1.3 X 10(5) nucleotides. Four weeks after treatment, these levels had declined to 1 adduct in 1.4 X 10(6) - 2.7 X 10(6) nucleotides. Substantial removal of DNA adducts occurred during the first 2 weeks after carcinogen application while adducts remaining thereafter underwent little or no repair between 2 and 4 weeks after treatment. These results raise the possibility that the persistent adducts occupy specific genomic sites in quiescent cells where they may not be amenable to repair because of localized conformational alterations of DNA or shielding by associated proteins.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benzo(a)pyrene; Benzopyrenes; Carcinogens; Cell Transformation, Neoplastic; DNA; DNA Repair; DNA, Neoplasm; Female; Isotope Labeling; Methylcholanthrene; Mice; Mice, Inbred BALB C; Phosphorus Radioisotopes; Polycyclic Compounds; Skin

Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5'-nucleotidase are suggested as modulators of hyaluronate synthesis.

Topics: Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Hyaluronic Acid; Phosphorus Radioisotopes; Teratoma; Uridine; Uridine Diphosphate N-Acetylgalactosamine; Uridine Diphosphate N-Acetylglucosamine

Pyridoxal [32P] phosphate was prepared using [gamma-32P] ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.

Topics: Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cell Transformation, Viral; Erythrocyte Membrane; Erythrocytes; Escherichia coli; Fibroblasts; Membrane Proteins; Neoplasm Proteins; Orthomyxoviridae; Phosphorus Radioisotopes; Phosphotransferases; Pyridoxal Phosphate; Pyridoxine; Schiff Bases; Viral Proteins

32P-Labeled phospholipids with specific activities up to 400 mCi/mmole as well as [32P]CDP-choline were prepared by cultivation of mouse fibroblasts or mouse Ehrlich ascites cells in the presence of [32P]orthophosphate. The method was also used to prepare [methyl-3H]choline-labeled glycerophospholipids from [3H]choline. The yields and the specific activities of the phospholipids were significantly lower when preparations of ox white blood cells were used.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cattle; Cell Line; Cell Transformation, Neoplastic; Chromatography, Thin Layer; Cytidine Diphosphate Choline; Erythrocytes; Fibroblasts; Humans; Isotope Labeling; Leukocytes; Mice; Phosphatidic Acids; Phosphatidylcholines; Phospholipids; Phosphorus Radioisotopes; Structure-Activity Relationship; Tritium

Topics: Agglutination; Animals; Blood Proteins; Cattle; Cell Count; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Contact Inhibition; Culture Media; Mice; Phosphates; Phosphorus Radioisotopes; Simian virus 40; Trypsin

Topics: Adenoviridae; Animals; Aviadenovirus; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Cricetinae; DNA; DNA, Neoplasm; DNA, Single-Stranded; DNA, Viral; Hot Temperature; Kidney; Nucleic Acid Denaturation; Phosphorus Radioisotopes; Skin

A rat type C virus spontaneously activated from the NRK (normal rat kidney) cell line was found to have two major size classes of viral RNA subunits sedimenting at 35 and 30 S. Virus-producing cells contained both RNA species, while normal "virus-free" rat cells contained primarily virus-specific 30S RNA species. A DNA transcript, specific for Kirsten sarcoma virus, prepared from virus activated in nonproducer BALB/c cells originally transformed by Kirsten sarcoma virus and rendered specific for the virus by absorption of sequences related to mouse helper virus hybridized only with the 30S RNA species of virus-producing rat cells and normal rat cells. These findings are consistent with the hypothesis that sarcoma-specific nucleic acid sequences in kirsten sarcoma virus emerged through a process that incorporated some portions of 30S RNA species from rat cells (either normal or virus-producing) into the Kirsten leukemia virus during passage in vivo of that virus. The virus designated M-MSV(RaLV), which originally derived from tumor induced by Moloney sarcoma virus (M-MSV) in rats, contained 35S RNA species of rat type C viruses and 30S RNA species specific for both rat and mouse viruses. It appears striking that for these two animal species, sarcoma-virus-specific information resides on a 30S subunit.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Zonal; DNA, Viral; Gammaretrovirus; Kidney; Mice; Molecular Weight; Moloney murine leukemia virus; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Rats; Retroviridae; RNA; RNA-Directed DNA Polymerase; RNA, Viral; Sarcoma, Experimental; Transcription, Genetic; Tritium

Topics: Adenoviridae; Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Chromatography; DNA, Viral; Endonucleases; Haplorhini; Humans; Hydroxyapatites; Kinetics; Mice; Molecular Weight; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Rats; RNA; RNA, Viral; Simian virus 40; Transcription, Genetic

Topics: Cell Line; Cell Transformation, Neoplastic; Chromatography, Paper; Cyclic P-Oxides; Glycerol; Inositol; Isotope Labeling; Lung; Phosphates; Phosphatidylinositols; Phosphorus Radioisotopes; Simian virus 40; Time Factors; Tritium

Topics: Animals; Avian Sarcoma Viruses; Cell Division; Cell Transformation, Neoplastic; Chick Embryo; Contact Inhibition; Culture Techniques; Cyclic AMP; Cyclic GMP; DNA; Fibroblasts; Insulin; Neuraminidase; Nucleotides, Cyclic; Phosphates; Phosphoproteins; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes; Protein Kinases; Stimulation, Chemical; Thymidine; Tritium

Isolated nuclei, prepared from myeloblasts of chicks infected with avian myeloblastosis virus, synthesize RNA sequences present in avian myeloblastosis viral RNA. These sequences are also formed during transcription of chromatin, isolated from myeloblasts, by DNA-dependent RNA polymerases purified from Escherichia coli or calfthymus. In the latter case, transcription is alpha-amanitin sensitive. Formation of hybrids between RNA and avian myeloblastosis virus DNA probes has been monitored by the combined use of ribonucleases A, T(1), and H, and ribonucleases specific for single strands.

Topics: Animals; Avian Leukosis Virus; Avian Myeloblastosis Virus; Bone Marrow Cells; Cell Nucleus; Cell Transformation, Neoplastic; Chick Embryo; Chromatin; DNA-Directed RNA Polymerases; DNA, Single-Stranded; Escherichia coli; In Vitro Techniques; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Ribonucleases; RNA, Viral; Templates, Genetic; Thymus Gland; Transcription, Genetic; Tritium; Virus Replication

Topics: Adenine Nucleotides; Animals; Autoradiography; Avian Sarcoma Viruses; Base Sequence; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Chick Embryo; Chromatography, Affinity; Culture Techniques; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Molecular Weight; Nucleotides; Oligonucleotides; Phosphorus Radioisotopes; Polynucleotides; Ribonucleases; RNA, Viral

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Edetic Acid; Fibroblasts; Gammaretrovirus; Glyceraldehyde-3-Phosphate Dehydrogenases; Mice; Mice, Inbred BALB C; Muscles; NAD; Phosphoglycerate Kinase; Phosphorus Radioisotopes; Rabbits; Time Factors; Trypsin

(32)P-Labeled SV40 DNA was treated sequentially with restricting endonucleases EcoRI and Hpa I, and the resulting four fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete SV40 DNA were measured in the presence of DNA extracted from the SVT2 line of SV40-transformed mouse cells. It was found that these cells contain about six copies of a segment of DNA which includes the early region of the SV40 genome, and about one copy of the late viral sequences. To map the region of the viral genome which is transcribed in SVT2 cells, separated strands of each of the four fragments were prepared and hybridized to total transformed cell RNA. Part of the E strands of the two DNA fragments (A and C) which span the early region of the SV40 genome were found to enter the hybrid.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Chromosome Mapping; Clone Cells; Diploidy; DNA, Viral; Endonucleases; Fibroblasts; Genes; Hydrolysis; Kinetics; Mice; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Nucleic Acid Renaturation; Phosphorus Radioisotopes; Polynucleotides; RNA, Viral; Simian virus 40

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Cell Line; Cell Transformation, Neoplastic; Enzyme Activation; Fibroblasts; Guanosine Triphosphate; Kidney; Kinetics; Leukemia Virus, Murine; Magnesium; Manganese; Moloney murine leukemia virus; Phosphorus Radioisotopes; Prostaglandins; Protein Binding; Rats; Species Specificity; Time Factors

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells; DNA, Neoplasm; Kinetics; Mice; Microwaves; Mitosis; Phosphorus; Phosphorus Radioisotopes; Radiation Effects; Temperature

Topics: Adenoviridae; Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chromatography; DNA; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Endonucleases; Fibroblasts; Genes; Genotype; Kinetics; Nucleic Acid Hybridization; Nucleic Acid Renaturation; Phosphorus Radioisotopes; Rats

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Cricetinae; Dimethyl Sulfoxide; Electrophoresis, Polyacrylamide Gel; Mice; Moloney murine leukemia virus; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Rats; RNA, Viral; Tritium

Topics: Animals; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Chromatography, Gel; Chromatography, Ion Exchange; Deoxyribonucleases; Deoxyribonucleotides; DNA, Neoplasm; Electrophoresis, Polyacrylamide Gel; Guanine Nucleotides; Isotope Labeling; Mice; Molecular Weight; Pancreas; Phenylalanine; Phosphorus Radioisotopes; Ribonucleases; Ribonucleotides; RNA, Neoplasm; RNA, Transfer; Tritium

Topics: Animals; Cell Division; Cell Fractionation; Cell Transformation, Neoplastic; Cells, Cultured; Centrifugation, Density Gradient; Chick Embryo; Deuterium; DNA-Directed RNA Polymerases; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Guanine Nucleotides; Phosphorus Radioisotopes; Polyribosomes; Ribonucleases; Ribosomes; RNA, Ribosomal; RNA, Viral; Sindbis Virus; Time Factors; Tritium; Viral Proteins

The genetic information contained in the Kirsten and Moloney strains of mammalian RNA-containing sarcoma viruses has been analyzed by RNA . (3)H-DNA hybridization. Kirsten sarcoma virus has been found to possess two distinct sets of nucleic acid sequences. One set of sequences is contained in murine type C helper virus, and the other set is contained in rat type C helper virus. Moloney sarcoma virus contains sequences of murine type C helper virus but not of rat type C helper virus. The results indicate that Kirsten sarcoma virus arose through a process of recombination between Kirsten murine leukemia virus and nucleic acid sequences found in rat cells. A model is suggested for the formation of transforming type C viruses involving the transduction of oncogenic information.

Topics: Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; DNA, Viral; Helper Viruses; Leukemia Virus, Murine; Mice; Models, Biological; Moloney murine leukemia virus; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Rats; Recombination, Genetic; Retroviridae; RNA-Directed DNA Polymerase; RNA, Viral; Transduction, Genetic; Tritium; Virus Cultivation

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell-Free System; Centrifugation, Density Gradient; DNA, Viral; Haplorhini; Leukemia Virus, Feline; Magnesium; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Retroviridae; Ribonucleases; RNA-Directed DNA Polymerase; RNA, Viral; Sarcoma, Experimental; Satellite Viruses; Skin; Templates, Genetic; Tritium; Virus Cultivation

Topics: Animals; Bacillus subtilis; Cattle; Cell Line; Cell Transformation, Neoplastic; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Paper; Coliphages; DNA; DNA Nucleotidyltransferases; DNA, Bacterial; Escherichia coli; Humans; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Magnesium; Molecular Weight; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Polynucleotide Ligases; Spectrophotometry, Ultraviolet; Thymus Gland; Tritium

Topics: Animals; Base Sequence; Cell Transformation, Neoplastic; Cells, Cultured; Chronic Disease; Electrophoresis, Paper; Electrophoresis, Polyacrylamide Gel; Molecular Conformation; Molecular Weight; Moloney murine leukemia virus; Phosphorus Radioisotopes; Rats; RNA, Neoplasm; RNA, Viral; Sarcoma, Experimental; Tritium; Ultracentrifugation

RNA from noninfectious virions produced by two established clonal lines of sarcoma positive-leukemia negative (S+L-)-transformed 3T3 cells has been characterized. RNA from virions or nucleoids of S+L--(C243) cells consisted of three to four sizes: +/-44 S (6%), 28 S (17%), 18 S (38%), and <18 S (39%). 28S virion RNA contained some virus-specific information demonstrable by RNA.DNA hybridization with a DNA probe derived from the RNA-directed DNA polymerase product of murine sarcoma-leukemia virus, while parallel studies indicated that 28S ribosomal RNA from ribosomal subunits of transformed and nontransformed 3T3 cells did not contain virus-specific information. In contrast to the S+L-(C243) virions, RNA from virions or nucleoids of S+L-(D56) cells consisted of five sizes: 80 S (6%), 68 S (8%), 56 S (5%), 28 S (28%), and <28 S (53%). Thermal dissociation studies suggested that this S+L- genome is comprised of 28S RNA subunits. From these studies we postulate that the 28S viral RNA is most probably the monomeric genome of S+L- virions.

Topics: Adenosine; Animals; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Clone Cells; DNA, Viral; Fibroblasts; Gammaretrovirus; Helper Viruses; Mice; Molecular Weight; Moloney murine leukemia virus; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Polynucleotides; Polyribosomes; Ribosomes; RNA, Ribosomal; RNA, Viral; Tritium; Uridine

Topics: Animals; Avian Sarcoma Viruses; Cell Fractionation; Cell Nucleus; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Chick Embryo; Culture Techniques; Cytoplasm; DNA, Viral; Fibroblasts; Glucose; Microscopy, Phase-Contrast; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Polyribosomes; RNA, Viral; Spectrophotometry; Time Factors; Tritium; Uridine

Separated strands of simian virus 40 (SV40) DNA fragments were used in hybridization experiments to study the RNA transcribed by Escherichia coli RNA polymerase from the chromatin of cells transformed by SV40. The template activity of chromatin of the transformed cell line 11A8 (mouse-embryo cells) examined is about 17% that of purified DNA, suggesting that most of the chromatin DNA is repressed by chromosomal proteins. The SV40-specific RNA present in the RNA transcribed in vitro from 11A8 chromatin hybridizes specifically with the minus strand of SV40 DNA. Little or no reaction occurs with the plus strand of viral DNA. The SV40-specific RNA transcribed in vitro from chromatin of transformed cells shares sequences with the RNA produced during the early phase of SV40 lytic infection, and is similar to that present in the 11A8 cell line in vivo. Although the influence of chromosomal proteins on this pattern of transcription was not definitely determined, preliminary evidence indicates that an asymmetric pattern of transcription may also occur when 11A8 DNA is transcribed by E. coli RNA polymerase.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Chromatin; DNA; DNA-Directed RNA Polymerases; DNA, Viral; Embryo, Mammalian; Escherichia coli; Fibroblasts; In Vitro Techniques; Kinetics; Liver; Mice; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Rats; RNA, Viral; Simian virus 40; Templates, Genetic; Transcription, Genetic

Topics: Animals; Biological Transport; Cell Line; Cell Transformation, Neoplastic; Fibroblasts; Glycerol; Isotope Labeling; Kinetics; Mice; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phosphorus Radioisotopes; Simian virus 40; Solubility; Time Factors; Tritium

Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Chromatography, Thin Layer; Cricetinae; Cytosine; Deoxycytidine; DNA; DNA, Mitochondrial; Escherichia coli; Kidney; L Cells; Methionine; Methylation; Methyltransferases; Mice; Phosphorus Radioisotopes; Polyomavirus; Subcellular Fractions; Tritium

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell-Free System; Ceramides; Choline; Chromatography, Thin Layer; Cytosine Nucleotides; Fibroblasts; Kinetics; Mice; Organophosphorus Compounds; Phosphatidylcholines; Phosphorus Radioisotopes; Simian virus 40; Sphingomyelins; Time Factors; Tritium