Jean Jackson

Transition series metals and sulfhydryl reagents induce the synthesis of four proteins in eukaryotic cells

Several transition series metals (copper, cadmium, zinc and mercury) and the sulfhydryl reagent, sodium arsenite, enhance the synthesis of specific proteins in chick embryo cells and in human foreskin cells in culture. The proteins are visible 1--3 h after exposure to concentrations ranging from 10 microM to 100 microM depending upon which reagent is used. These proteins comigrate on acrylamide gel electropherograms with the proteins induced by two copper-chelating drugs, kethoxal bis(thiosemicarbazone) and disulfiram, and by heat shock. However, these proteins migrate in a significantly different manner than do the canavanine-enhanced proteins. The four proteins induced in chick embryo cells are distinct from one another as determined by partial proteolytic mapping. Simlarly, the three proteins induced in human cells are distinct. However, the 100-kilodalton and the 70-kilodalton proteins from chick and from human cells appear to be related as judged by this mapping procedure. The 70 kilodalton protein enhanced by kethoxal bis(thiosemicarbazone), disulfiram, arsenite and heat shock have a high degree of similarity according to this technique. The arsenite and canavanine-enhanced 100-kilodalton proteins are related as are the arsenite-enhanced 70-kilodalton and the canavanine-enhanced 75-kilodalton proteins. The canavanine-enhanced 30 kilodalton protein resembles the arsenite-enhanced 25-kilodalton protein rather than the 35-kilodalton species. In view of these findings, it appears that a variety of treatments, namely, chelating drugs, transition series metals, sulfhydryl reagents, heat shock, and amino acid analogous can induced similar, if not identical, proteins in eukaryotic cells.

The low molecular weight RNAs of Rous sarcoma virus

Abstract Chromatography of Rous sarcoma virus RNA on methylated albumin-kieselguhr has allowed the identification of four classes of intravirion RNA: a substantial amount of 4 S RNA, a discrete 7 S component, small quantities of 18 and 28 S RNA, and the 70 S RNA of the viral genome. The 4 S RNAs of the virus and its host cell have identical electrophoretic mobilities in 10% polyacrylamide gels and are methylated to the same extent. However, significant differences in nucleotide compositions are detectable. The virus apparently does not contain RNA corresponding to any of the other low molecular weight species of cellular RNA. The data are in accord with previous suggestions that RNA tumor viruses contain tRNA acquired from the host cell during assembly, but also indicate at least minor differences in composition between the cellular and viral 4 S RNA populations. The results of reconstruction experiments suggest that the viral 4 S RNA is not simply a contaminant derived from cellular debris.

Metal binding drugs induce synthesis of four proteins in normal cells.

The synthesis of four proteins in chick embryo cells in culture is induced by exposure to several metal binding drugs and several cations. We reported previously that two chelating agents, kethoxal bis(thiosemicarbazone) and disulfiram induce these proteins. We now extend these findings to include 8-hydroxyquinoline, ortho-phenanthroline, and thiosemicarbazide. The non-chelating analogues of these latter three compounds; 4-hydroxyquinoline, metaphenanthroline, and semicarbazide, do not induce. Other chelating agents, such as thenoyl trifluoroacetone, mercaptopyridine N-oxide, mercaptobenzothiazole, and methimazole induce. However, not all chelators induce since EDTA, penicillamine, α,α-dipyridyl, and several others are ineffective.Several cations, such as copper, zinc, cadmium, and mercuric ions induce, but cobalt, nickel, manganese, iron, lead, and platinum do not. The anions arsenite and arsenate induce, but bromide, dichromate, fluoride, metabisulfite, molybdate, nitrite, permanganate, phosphate, sulfite, and sulfate ions do not.The chelating agents that induce the proteins are ionophores for64Cu. The chelating agents that do not induce, such as EDTA, penicillamine, and the nonchelating analogs of the inducers, are not ionophores for copper.Since arsenite induces but is not a copper ionophore, we hypothesize that the common mechanism for both these compounds and the inducing cations is an interaction with a sulfhydryl group site.

Hydroxyquinolines Inhibit Ribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase and Inactivate Rous Sarcoma Virus and Herpes Simplex Virus

8-Hydroxyquinoline and several of its derivatives inactivate the transforming ability of Rous sarcoma virus and inhibit its ribonucleic acid-dependent deoxyribonucleic acid polymerase activity. The copper complex of these metal-binding ligands is as active as the free ligand. The activity of the 8-hydroxyquinolines is approximately 50-fold more effective than another group of metal-binding compounds that we have tested, the thiosemicarbazones. In contrast to the potency of the 8-hydroxyquinolines to inactivate Rous sarcoma virus, no intracellular inhibition of transformation could be demonstrated at a concentration that did not affect the growth and appearance of the cells. Cellular deoxyribonucleic acid synthesis was inhibited to a greater extent than was ribonucleic acid or protein synthesis. The phenomenon of “concentration quenching” was observed with high concentrations of drug, causing less inhibition of deoxyribonucleic acid synthesis than was observed with lower concentrations. Herpes simplex virus type 1 was inactivated also by the 8-hydroxyquinolines and their copper complexes. No intracellular inhibition of plaque formation was observed. Treatment with 8-hydroxyquinoline sulfate had no effect on the resolution of herpetic keratitis in rabbits. Some 8-hydroxyquinolines bind to deoxyribonucleic acid in the presence of copper, a phenomenon that may be important in their antiviral activity.

Effect of antabuse (disulfiram) on Rous sarcoma virus and on eukaryotic cells

Antabuse (disulfiram) is widely used in the treatment of chronic alcoholism. We have examined the effect of this drug on malignant transformation by Rous sarcoma virus, on eukaryotic cell synthesis, and on nucleic acid binding. It was found that: (1) Disulfiram inhibits the activity of the RNA dependent DNA polymerase of Rous sarcoma virus and inactivates the ability of the virus to malignantly transform chick embryo cells. The monomer of disulfiram, diethyldithiocarbamate does not affect the virus. (2) Disulfiram induced the synthesis of four proteins in normal chick embryo and human foreskin cells. The monomer diethyldithiocarbamate, induced these proteins also. Cellular DNA synthesis is more sensitive to disulfiram than are RNA and protein synthesis. (3) Disulfiram binds to neither DNA or RNA in the presence or absence of copper. However, diethyldithiocarbamate in the presence of, but not in the absence of, copper binds to HeLa cell DNA and to Rous sarcoma virus 70 S genome RNA. These results indicate that this compound, which causes no symptoms in people who do not consume alcohol, may have significant effects on a cellular level.

A gene related to the proto-oncogene fps/fes is expressed at diverse times during the life cycle of Drosophila melanogaster.

The proto-oncogene fps/fes encodes a distinctive type of protein-tyrosine kinase. We identified a Drosophila gene (dfps85D) whose product resembles the proteins encoded by vertebrate fps/fes and the closely related gene fer. dfps85D is located at chromosomal position 85D10-13 and is unlikely to correspond to any previously defined genetic locus in Drosophila melanogaster. Expression of the gene is entirely zygotic in origin and occurs throughout the life cycle. But hybridization in situ revealed that the pattern of expression is specialized and evolves in a provocative manner. The most notable feature of expression is the diversity of developmental periods, tissues, and cells in which it occurs. In some tissues, expression is transient; in others, it is continuous. Expression occurs in both mitotic and terminally differentiated tissue and, at various times in development, is prominent in imaginal disks, gut, muscle, testes, ovaries, retina, and other neural tissues. It appears that the use of dfps85D is more diversified than that of other Drosophila protein-tyrosine kinases reported to date and contrasts sharply with the restricted expression of fps itself in vertebrates. The detailed description of expression provided here will help guide the search for mutants in dfps85D.

Induction of four proteins in eukaryotic cells by kethoxal bis(thiosemicarbazone)

Kethoxal bis(thiosemicarbazone) induces the synthesis of four proteins (100 000, 70 000, 35 000 and 25 000 daltons) in normal chick embryo cells. The 70 000 dalton species is produced at the fastest rate 2 hr after exposure to the compound. Pulse-chase experiments revealed neither precursors nor products of these proteins and both actinomycin and cycloheximide inhibited their synthesis. Neither of the two substituents of the inducer, kethoxal or thiosemicarbazide, were active. The four proteins were induced in several other species, but human cells produced only three proteins (100 000, 70 000 and a different 30 000 dalton form).

INACTIVATION AND INHIBITION OF ROUS SARCOMA VIRUS BY COPPER‐BINDING LIGANDS: THIOSEMICARBAZONES, 8‐HYDROXYQUINOLINES, AND ISONICOTINIC ACID HYDRAZIDE *

We have shown that three types of copper-binding ligands, thiosemicarbazones, 8-hydroxyquinolines, and isonicotinic acid hydrazide and their copper complexes, inactivate the transforming ability of RSV and inhibit its RNA-dependent DNA polymerases. Three other compounds, 2-pyridine thiosemicarbazone, 1-formyl isoquinoline thiosemicarbazone, and diphenyl thiocarbazone inhibit transformation by RSV intracellularly. Most but not all of these compounds bind to nucleic acids in the presence of copper, which may be important in their mode of action.

Effect of isonicotinic acid hydrazide-copper complex on Rous sarcoma virus and its genome RNA

The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed.

Interaction of psoralen derivatives with the RNA genome of Rous sarcoma virus

Abstract We have examined the interaction of several psoralen derivatives with the RNA genome of Rous sarcoma virus (RSV). In the presence of long-wavelength uv light, 4,5′,8-trimethylpsoralen (trioxsalen) and its 4′-hydroxymethyl (HMT) and 4′-aminomethyl (AMT) derivatives all efficiently crosslinked the subunits of the RSV 70 S RNA complex into a nondissociable state. These psoralen derivatives also were able to penetrate virions and crosslink the RNA complex in situ. The ability of psoralen to crosslink the subunits of viral RNA in situ is consistent with the presumption that the RSV genome exists as a complex within the virion and suggests an approach for a detailed examination of regions of secondary structure. The addition of psoralen adducts to viral RNA correlated with a loss of virus infectivity and a decrease in the amount and length of viral DNA synthesized in vitro and in vivo. Our results indicate that the RSV genome contains a significant amount of secondary structure that is reactive with psoralen compounds and that the addition of HMT to viral RNA causes premature chain termination during viral DNA synthesis.