RESUMO
The carcinogen-treated cockerel is a model for studying the early stages of arteriosclerotic plaque development. Carcinogen administration accelerates arteriosclerotic plaque development in cockerels, and transforming elements are present in DNA from advanced human plaques. In this study, we asked whether transforming elements could also be detected at early stages of plaque development in cockerels. NIH3T3 cells were transfected with DNA from plaques isolated from carcinogen-treated cockerels and from the healthy arterial wall underlying the plaques. Approximately 5 x 10(6) cells from each group were injected into nude mice. Tumors appeared in five of five mice in the plaque DNA group; no tumors appeared in mice from the healthy arterial wall group. All five plaque DNA-associated tumors hybridized to a cockerel genomic probe. Eight cockerel-specific bands were identified in EcoRI digests of first-round (primary) tumors. DNA from a primary tumor was tested in a second round of transfection. Five of five mice developed tumors after injection with these secondary transformants. All second-round tumors contained cockerel DNA, and a prominent cockerel-specific band (greater than 28 kb) was seen in EcoRI digests of all second-round tumors. In addition, a 5.2-kb band appeared prominently in one of five second-round tumors. No evidence was found for activation of the oncogenes Ha-ras, Ki-ras, src, or myc in the plaque-associated tumors. Similarly, DNA from plaque-associated tumors did not hybridize to probes for Marek disease virus, herpes simplex virus 1, or reverse transcriptase, suggesting that neither herpesviruses nor retroviruses are involved in the transforming activity of plaque DNA. These results indicate that transforming elements are a general property of arteriosclerotic plaques and are detectable in plaques of young animals.
Assuntos
Arteriosclerose/genética , Oncogenes/genética , 9,10-Dimetil-1,2-benzantraceno , Animais , Arteriosclerose/induzido quimicamente , Linhagem Celular Transformada , Galinhas , DNA/genética , DNA de Neoplasias/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Nus , Neoplasias Experimentais/genética , Hibridização de Ácido Nucleico , Transfecção/genéticaRESUMO
The proliferation of vascular smooth muscle cells (SMC) is critical to atherosclerotic plaque formation. The monoclonal hypothesis proposes that the stimulus for this SMC proliferation is a mutational event. Here we describe a procedure for growing human plaque smooth muscle cells (p-SMC) in culture. We show that p-SMCs derived from two patients differ from SMC cultured from normal vascular tissue in expression of the protooncogene myc. One p-SMC strain was extensively characterized; these diploid, karyotypically normal cells have a finite life span in culture. Ultrastructural examination revealed two populations, one with classic contractile SMC appearance, the other, modulated to a synthetic state. Northern blotting showed a 2- to 6-fold and a 6- to 11-fold enhanced expression of myc by p-SMC, compared to SMC derived from healthy human aorta (HA-SMC) and saphenous vein (HV-SMC), respectively. In contrast, the p-SMC and HV-SMC expressed similar levels of message for the genes N-myc, L-myc, Ha-ras, fos, sis, myb, LDL receptor, EGF receptor, IGF I receptor, IGF II, and HMG CoA reductase. Finally, although p-SMCs are not tumorigenic, DNA isolated from these cells is positive in the transfection-nude mouse tumor assay. Myc, however, does not appear to be the transforming gene because no newly introduced human myc gene was detected in the p-SMC-associated nude mouse tumor. Thus human atherosclerotic p-SMCs possess both an activated myc gene and a transforming gene that is retained throughout many cell passages.
Assuntos
Arteriosclerose/genética , Músculo Liso Vascular/fisiopatologia , Oncogenes , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Transformação Celular Neoplásica , Células Cultivadas , Humanos , Camundongos , Camundongos Nus , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , TransfecçãoRESUMO
The major finding of this work is that there are specific restriction enzyme inhibitors present in "purified" tumor DNA which cause partial digestion patterns when tumor DNA is digested by standard procedures with any of three commonly employed restriction enzymes (HindIII, KpnI, XbaI). These aberrant patterns are not seen when DNA of cell lines derived from these tumors is digested. Thus, when working with tumor DNA these restriction enzymes should be used with caution.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA de Neoplasias/metabolismo , Animais , Linhagem Celular Transformada , Doença das Coronárias/genética , Enzimas de Restrição do DNA/antagonistas & inibidores , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias da Bexiga Urinária/genéticaRESUMO
The human erythrocyte membrane is an efficient enhancer of both high (CA II) and low (CA I) activity isozymes of red blood cell carbonic anhydrase. The presence of membrane increased CO2 hydration catalyzed by bovine CA II 1.6-fold, human CA II 3.5-fold, and human CA I 1.6-fold. With the high activity CA isozymes, maximal stimulation was observed in the presence of 1-3 micrograms membrane protein/ml. The Vmax for bovine CA II (4 nM) rose from 0.302 to 0.839 mM/s, while that for human CA II (6 nM) increased from 0.113 to 0.414 mM/s in the absence and presence of membrane, respectively. The apparent Km for CO2 increased from 13.2 to 51.2 mM for bovine CA II, and from 6.5 to 38.5 mM for human CA II. Mixtures of membrane plus enzyme, upon centrifugation through linear sucrose density gradients, displayed enhanced Ca activity only in membrane-containing gradient fractions, verifying the stimulatory ability of membranes on enzyme activity and indicating tight and stable complex formation. Membrane enhancement of CA activity appears to be a general phenomenon in that mouse hepatocyte membranes also stimulated CA activity, although less efficiently than erythrocyte membranes. Of the many soluble putative effectors assayed, only imidazole enhanced CA II activity to an extent comparable with erythrocyte membranes; imidazole did not, however, stimulate the activity of human CA I. The data are consistent with a model of CA II activation by membrane association that may effect a distortion of the enzyme conformation in such a way as to facilitate intra- and/or intermolecular proton transfer between membrane-bound and enzyme-bound proton shuttling residues (perhaps the imidazole moiety of histidine) and the Zn-bound hydroxide at the catalytic site of the enzyme.
Assuntos
Anidrases Carbônicas/sangue , Membrana Eritrocítica/fisiologia , Eritrócitos/enzimologia , Isoenzimas/sangue , Aminoácidos/farmacologia , Animais , Bovinos , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Cinética , Fígado/metabolismo , Masculino , Lipídeos de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Peptídeos/farmacologia , Fosfolipídeos/farmacologia , Ligação ProteicaRESUMO
Apo-A-I, the major protein component of high density lipoproteins, appears intracellularly as an intermediate precursor (pro-apo-A-I) with a hexapeptide extension (RHFWQQ) at its amino terminus. Proteolytic processing of pro-apo-A-I to apo-A-I has been shown to occur extracellularly in cell and organ cultures from rat and human tissues. Recently, however, intracellular conversion has been detected in chickens. To determine what distinguishes and regulates these two processing methods, the proteolytic processing and secretion of apo-A-I was studied by metabolic labeling in chick hepatocytes and in Hep-G2 cells (derived from a human hepatocellular carcinoma). The proportions of intracellular and secreted pro-apo-A-I and apo-A-I were measured by sequencing NH2-terminal portions of the proteins and determining the location of radio-labeled amino acids. Chick hepatocytes cultured in the absence of hormones or fetal bovine serum secreted primarily processed apo-A-I (83%). In the presence of serum these cells secreted only pro-apo-A-I, whereas incubation with a combination of hormones (insulin, triiodothyronine, dexamethasone) resulted in secretion of a nearly equal mixture of the pro- and processed forms of the protein. In contrast, Hep-G2 cells, maintained in the absence of serum, secreted only pro-apo-A-I; when grown in the presence of serum these cells secreted a mixture of pro- and processed apo-A-I. Under conditions in which chick hepatocytes and Hep-G2 cells secreted both forms of the protein, a mixture of pro- and processed apo-A-I was also found intracellularly; when only the pro-form was secreted, the cells likewise contained only pro-apo-A-I. Under all the above conditions, the secreted apo-A-I exhibited similar isoform patterns in two-dimensional gel electrophoresis. These data show that both chick hepatocytes and human hepatoma cells are capable of intracellularly processing pro-apo-A-I to apo-A-I, and that the extent of intracellular processing is controlled by the cell's hormonal environment.