Inactivation of SV40 replication by derivatives of benzo[a]pyrene.

When African green monkey kidney cell lines, infected with simian virus 40, were exposed to benzo[a]pyrene-7,8-dihydrodiol or $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, inhibition of progeny virus formation was observed. Alkylation of SV40 DNA with $\text{anti}$-BPDE inhibits the infectivity of this viral DNA; however, the inactivation does not follow a single-hit mechanism. Studies on [³H]thymidine incorporation indicate that SV40 DNA synthesis is markedly impaired for the first 12 hours following BPDE treatment; 24 to 36 hours later, however, SV40 DNA synthesis is almost normal. These data suggest that the inhibition of SV40 DNA synthesis by BP derivatives is reversible and that the observed reduction in viral titer requires some other explanation.     

The second large simian virus 40 T-antigen exon contains the information for maximal cell transformation

Microinjection of early simian virus 40 (SV40) DNA fragments has shown that maximal transformation of rat cells (Ref 52) is a property of the second SV40 T-antigen exon. Expression of this particular T-antigen region was obtained by coinjection of the $\text{Taq}\text{Bam}$ DNA fragment with the early promoter/enhancer $\text{Hpa}\text{II}\text{Bgl}\text{I}$ fragment. Microinjection of the DNA fragment mixture induced two categories of transformants; namely, maximally and minimally transformed cells. The maximally transformed cells synthesize two $\text{Taq}\text{Bam}$-specific polypeptides, and the minimally transformed cells only the lower molecular weight form. Both types of transformants contain the cellular p52 protein at high concentrations. Furthermore, maximal transformation of Ref 52 cells requires the carboxy terminus of the T-antigen. Cells transformed by microinjection of the SV40 Pst A-fragment display different parameters of maximally transformed cells but not anchorage-independent growth.     

Diverse directional changes of cGMP relative to cAMP in E. coli.

The relationships of the changes of cAMP and cGMP concentrations in $\text{E. coli}$ varied as a function of experimental conditions. (1) Cells starved for carbon source for a short time period had high cAMP and low cGMP concentrations. Addition of carbon source (succinate, glucose or α-methyl glucoside) led to a decrease in cAMP and an increase in cGMP (bi-directional change). (2) Washed cells starved for glucose for long time periods had low cAMP levels which did not change on glucose addition. Addition of succinate or glucose to such cells led to a transient increase in cGMP levels (uncoupled change). The cGMP concentration peaked at 15 minutes or 1 hour after glucose or succinate addition, respectively. (3) Sham shift-up experiments (addition of α-methyl glucoside to cultures growing in succinate) in $\text{E. coli}$ 1100 and CA 8000 showed decreases in cGMP levels in both strains; however, cAMP levels increased in the former (bi-directional change) and decreased in the latter (unidirectional change).     

The fate of SV40 DNA in infected cells after treatment with benzo(a)pyrene diol epoxide

Treatment of SV40-infected monkey kidney cells with $\text{anti}$-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide results in the alkylation of viral DNA. The specific infectivity of viral DNA isolated from diol epoxide-treated cells is markedly lower than that of viral DNA from untreated cells. Within 5–6 hours following treatment, approximately half of the alkylated viral DNA disappears; during this same period, single-stranded regions in SV40 DNA are detected. These data indicate that, in infected cells, a significant amount of the alkylated viral DNA is removed from the cellular DNA pool while the remainder stays intact. Whether BP-modified SV40 DNA undergoes repair is still uncertain.     

Synthesis of simian virus 40 DNA in isolated nuclei

The presence or absence of calcium ions during the isolation of nuclei from SV40-infected African green monkey kidney cells significantly affects the size of SV40 DNA synthesized $\text{in vitro}$. When Ca⁺⁺ is present during the nuclear isolation procedure, the 3–7S fragments of SV40 DNA synthesized $\text{in vitro}$ mature into long chains; in the absence of Ca⁺⁺ they do not.     

Prostaglandin E2 rather than lymphocyte-activating factor produced by activated human mononuclear cells stimulates increases in murine thymocyte cAMP

Culture supernatants (SUPS) of endotoxin (LPS)-activated human mononuclear cells (MNL) stimulated greater production of cAMP by thymocytes than by spleen cells of C3H/HeJ or nude ($\text{nu}\text{nu}$) mice. Similarly, the addition of prostaglandin E₂ (PGE₂) stimulated higher levels of cAMP in thymocytes and progressively lower levels in spleen cells from C3H/HeJ mice and $\text{nu}\text{nu}$ spleen cells, respectively. Partial purification on Bio-Gel P100 of the LPS-induced MNL SUPS yielded peaks of thymocyte proliferative activity characteristic of lymphocyte activation factor (LAF) but these fractions failed to stimulate cAMP levels in thymocytes. Moreover, MNL SUPS induced with LPS in the presence of indomethacin retained their LAF activity but no longer increased thymocyte cAMP levels. Radioimmunoassay of the SUPS for PGE₂ revealed significantly higher levels of PGE₂ in the media of those MNL cultures stimulated by LPS than when stimulated by phorbol myristic acetate, phytohemagglutin, or extracted cell wall fraction of Actinomyces viscosus. Thus, PGE₂ is produced by human MNL and may exert considerable immunoregulatory effects mediated by elevation of lymphocyte cAMP levels.     

The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells.

Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O₂ oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from ¹⁴C/³H leucine studies suggest that MSH may cause increased $\text{de}$$\text{novo}$ synthesis of tyrosinase.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

Mechanism of glycogenolytic action of cycloheximide in rat liver

Cycloheximide (0.1 to 0.2 m$\text{M}$) increases cAMP concentration 3 to 4-fold in isolated rat liver slices $\text{in vitro}$. This increase in cAMP concentration parallels an increase in phosphorylase activity. When cycloheximide, at a concentration used to inhibit protein synthesis (1 to 2 μg/g body weight), is administered to whole animals, phosphorylase is activated up to 13-fold by 6 hours. This leads to almost complete depletion of liver glycogen (from about 40 mg/g liver to 0.4 mg/g liver).     

Induction of thymidylate synthetase activity in Tetrahymena by cyclic guanosine monophosphate.

The induction of TMP synthetase activity in Tetrahymena pyriformis depended upon growth conditions. Enzymatic activity was low in cells grown in complex medium, and was high in cells grown in, or shifted to, defined medium. TMP synthetase activity rose 5 hours after the shift from complex to defined medium using uracil as the pyrimidine source. The time of induction was decreased to $\text{3}\text{1}\text{2}$ hours using dUMP as the pyrimidine source. cGMP or its dibutyryl derivative, but not cAMP, caused the induction of TMP synthetase activity in cells grown in complex medium. Caffeine, but not theophylline, mimicked the cGMP response. cAMP decreased both the cGMP and caffeine mediated increases in TMP synthetase activity. This is the first demonstration of an effect of cGMP on induction of an enzyme of pyrimidine metabolism in any cellular system.     

Measurement of cyclic 3',5'-adenosine monophosphate synthesis in growing Escherichia coli.

The net synthesis of cAMP by an adenine auxotroph of $\text{Escherichia coli}$ was measured by assaying the incorporation of tritium from [³H]-adenine into cyclic [³H] AMP during exponential growth. Synthesis of cAMP ceased abruptly when glucose was added to cells growing in glycerol and then recovered to an intermediate rate of synthesis after 0.5–1.0 generation. Cyclic AMP appeared to be synthesized from a precursor pool that turned over more rapidly than total cellular ATP. The rates of cAMP synthesis measured by this technique are compatible with the cellular levels of cAMP previously measured in this strain(3).     

DNA strand scission by the antitumor protein neocarzinostatin

The antibiotic protein, neocarzinostatin, induces the scission of DNA strands $\text{in vivo}$ and $\text{in vitro}$. HeLa cell DNA prelabelled with [¹⁴C] thymidine is cut into large pieces with a peak at 80–90S when cells are incubated with 0.5 to 5.0 μg/ml of highly purified neocarzinostatin. Incubation of the antibiotic (0.5 μg/ml) with [³H] SV40 DNA in the presence of 2-mercaptoethanol results in the conversion of superhelical DNA I to nicked circular duplex DNA II. At high levels of drug, smaller fragments of linear DNA are produced. Strand breaks are detected in both neutral and alkaline sucrose gradients, indicating that drug susceptibility is not due to alkali-labile bonds.     

Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum

Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum. cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1?2}\text{s}$) and a class of slow-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15?150}\text{s}$). Exposure of D. discoideum cells to 1 mM cAMP for 30 min induces a reduction of cAMP binding (down-regulation; Klein, C. and Juliani, M.H. (1977) Cell 10, 329–335). The number of fast-dissociating sites was reduced by 80–90% in down-regulated cells. These sites are composed of two forms with high and low affinity which interconvert during the binding reaction. In down-regulated cells this transition still occurred in the residual sites. The accumulation of cellular cAMP levels induced by a saturating stimulus decreased by 80–90%. The number of slow-dissociating sites was not significantly reduced in down-regulated cells, but their affinity decreased about 10-fold. The accumulation of cellular cGMP levels induced by a saturating stimulus was not decreased; however, about 20-fold higher cAMP concentrations were required to induce the same response. These results demonstrate that the cAMP transduction pathways to adenylate cyclase and guanylate cyclase are down-regulated differently. Furthermore, the results suggest that the fast-dissociating sites are involved in the activation of adenylate cyclase, while the slow-dissociating sites are coupled to guanylate cyclase.     

The identification of a serum viability factor for SV3T3 cells as biotin and its possible relationship to the maintenance of Krebs cycle activity

A low-molecular weight-factor (“Peak III”) from calf serum, which enhances the viability (and hence growth) of simian virus 40-transformed 3T3 (SV3T3), but not 3T3, cells when grown in low-serum (0.15–0.30%, $\text{v}\text{v}$) Dulbecco's modified Eagle's medium, has been identified as the vitamin biotin. The extraction procedure involved acidification of the serum to pH 4.5, boiling, ultrafiltration of the supernatant through a Pellicon membrane, and Sephadex G-25 chromatography. Peak III was identified as biotin for the following reasons. (i) The viability/growth activity was completely retained on an avidin-Sepharose but not a Sepharose column. (ii) Peak III preparations contained a compound which reacted with the cyclic ureide-specific reagent, p-dimethyl-aminocinnamaldehyde, and which migrated on thin-layer chromatography with the same R_f as biotin. (iii) Peak III and biotin had the same biological effects on SV3T3 cells, including a reduction in the number of dead cells, a lowering of the amount of lactic acid accumulated, and a synergism with iron in stimulating growth. (iv) They were not additive in their effects at saturating doses. To test the hypothesis that at least part of the biotin viability/growth effect may be due to the maintenance of Krebs cycle intermediate levels through the activation of pyruvate carboxylase, Krebs cycle intermediates were added singly to cells in low-serum medium without biotin. Malate, citrate, isocitrate, and fumarate (but not oxaloacetate, α-ketoglutarate, and succinate) were growth stimulatory for SV3T3 but not 3T3 cells. When added in combinations they were no more effective than alone.     

Light-induced changes in cAMP levels in Limulus photoreceptors

Illumination reduces cAMP levels about 3-fold in $\text{Limulus}$ photoreceptors. Cyclic GMP levels are not significantly changed under identical conditions. In addition, the cAMP content in dark-adapted photoreceptors is about 4-fold the content of cGMP. It is proposed that cAMP may be involved in the regulation of the metabolism or function of photoreceptor systems which contain the photopigment in the transducing surface membrane.     

Structural requirements of quassinoids for the inhibition of cell transformation

The effects of eleven quassinoids on Rous sarcoma virus induced cell transformation and on growth of normal cells were examined. At concentrations of 0.15-1 μg/ml they inhibited foci formation (76–99 %) without toxic effects on normal cells. The most active compounds also affected virus production by transformed cells. In intact normal and transformed cells, protein and DNA synthesis was equally affected after 3 hours of exposure to quassinoids of both cell types. RNA synthesis was not inhibited. This study has shown that the structural requirement of a C-15 ester in the quassinoids for antileukemic activity $\text{in vitro}$ and $\text{in vivo}$ is not essential for their antitransforming activity.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Isolation and characterization of cyclic AMP-resistant variants from a functional adrenal cortical cell line.

We have isolated a number of cyclic AMP-resistant cell lines and characterized two of them, 3B4 and 10F2s, from a functional adrenal cortical cell line, Y-1. At seeding densities above $\text{100}\text{cells}\text{10}\text{cm}$ diameter plates, the variant cells are resistant in their morphological change, plating efficiency and growth to the normal effects of dibutyryl cyclic AMP (db-cAMP). At lower seeding densities, 3B4 and 10F2 have retained a slight sensitivity to db-cAMP in their plating efficiency and in their morphology. Studies with various nucleotides and cAMP analogues show that the inhibitory effects of db-cAMP on the growth and morphology of Y-1 cells are not due to degradation products of db-cAMP. There is loss of response to ACTH in the variant cell lines such that there are no effects of ACTH on plating efficiency, growth, morphology, steroidogenesis and cAMP excretion. In addition, the variant cell lines show lowered activities of the cAMP binding receptor and cAMP-dependent protein kinase. Preliminary studies indicate that in Y-1, cAMP markedly reduces protein phosphorylation, and it inhibits phosphate uptake. In the variants, the protein phosphorylation and phosphate uptake are maintained even in the presence of db-cAMP. The maintenance of phosphorylation in the presence of db-cAMP may play an important role in the ability of the cells to survive in high concentrations of db-cAMP. The variant cell lines can be stimulated by db-cAMP to increase steroidogenesis, although the stimulated levels of steroidogenesis in 3B4 and 10F2 are less than those in Y-1. The variant phenotype is stable in vitro and in vivo.     

Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.     

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel Mytilus edulis.

Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel $\text{Mytilus}$$\text{edulis}$. In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10^?6M to 10^?4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of $\text{Mytilus}$$\text{edulis}$ is mediated through a cAMP second messenger system.