Cellular Actinomycin

In addition to receptor-mediated apoptosis (see 15.5), there is another main pathway activated by various forms of cellular stress. Examples of stress effects that can induce apoptosis are y- and UV-radiation, treatment with cytotoxic drugs such as actinomycin D and removal of cytokines. As a consequence of stress, procaspase 8 is activated by a... 

Biedler, J.L. and Riehm, H. (1970) Cellular resistance to actinomycin D in Chinese hamster cells in vitro, cross-resistance, radioautographic, and cytogenetic studies. Cancer Research, 30 (4), 1174-1184. 

Actinomycin D is a peptide antibiotic, produced by Streptomyces parvulu. It interacts with cellular DNA and inhibits the replication of mammalian viruses that depend on cellular functions, e.g., rabies virus [18]. Mithramycin is a related compound that inhibits influenza and pseudorabies viruses probably due to inhibition of host cell RNA polymerase II [18]. 

Auxins are also involved in so-called long term responses which may be more directly gene-mediated responses. Auxins stimulate and sustain mRNA and protein synthesis to form enzymes that catalyze the production of cell wall materials, sugars, and other cellular components. Applied lAA has induced mRNA and protein synthesis in Avena coleoptiles (16), yeast cells (17), pea stem sections (18), and bean endocarp (19). Artichoke tuber disks responded to exogenously applied lAA with growth and a substantial amount of new RNA and protein synthesis. When the metabolic inhibitors actinomycin D and 8-azaguanine were added simultaneously with lAA, effects of the auxin were negated (20). 

Infection with picornaviruses results in a strong (though selective) inhibition of the cellular ENA synthesis (5, 4> 85> 84), and the appearance of a new, virus-induced ENA synthesizing activity. The latter was soon shown to be insensitive to the action of Actinomycin D (5), an antibiotic which prevent DNA-dependent ENA synthesis by intercalating in the dG-dC sequences of the template MA (6, 7) These observations suggested the possibility of treating infected cultures with the antibiotic in order to suppress host-cell activity and measxiring the incorporation of radioactive precursors into viral ENA. Since the precursors added to the medium do not equilibrate instantaneously with the intracellular pool of nucleotides, a correction for this fact must be introduced, at least for the very early times. 

Radioactive RF has been used to infect cells and search for the modifications of the parental molecules in each sub-cellular compartment. Such studies revealed that soon after infection with RF, the input d-s molecules are found in the cytoplasm as part of a Replicative-Intermediate-like structure (18). Treatment of the host-cell with interferon abolishes the infectivity of RF, but does not prevent the intracellular processing of RF into RI. In contrast, exposure of the cells to Actinomycin D inhibits the infectivity of RF and the intracellular transition to RI as well (36). As it was found that a cellular RNA polymerase specifically binds to RF (37)> it was postulated that RF served as abnormal template for a cellular RNA polymerase to transcribe the first viral messenger (18, 57). 

The cellular target of the 1,25-hydroxylated derivative is unknown. It is still debated whether the hormone acts at the mucosal or the serosal site of the intestinal cell. A number of observations have suggested that the metabolite might stimulate calcium absorption by modifying gene expression e.g., the 7 hour time lag between metabolite administration and the increase in calcium absorption, and the intranuclear localization of the metabolite. Vitamin D is known to induce the formation of the calcium binding protein and the calcium-sensitive ATPase. However, this view must be reconciled with the fact that actinomycin D does not interfere with calcium absorption while it is known to block the conversion of the 25-hydroxylate to the 1,25-hydroxylate derivative. 

A stimulus which alters the steady-state level of an endogenous cellular component may do so by influencing its rate of synthesis, its rate of break-down, or both. When administered to intact animals, phenobarbital or 3-methylcholanthrene increase (20-50%) the steady-state level of microsomal protein. Similarly, micro-somes from animals pretreated with phenobarbital or 3-methylcholanthrene incorporate radioactive amino acids into protein more rapidly than microsomes from control animals and this effect is blocked by co-administration of actinomycin-D. It was therefore assumed that the increased levels of microsomal protein and enzyme activity after inducers were the result of enhanced synthesis. However, turnover studies have revealed that phenobarbital in particular has a profound effect upon microsomal protein catabolism. Proteins of the endoplasmic reticulum were labelled by injection of radioactive amino acids and the rate at which radioactivity disappeared from the microsomes was compared in control and phenobarbital-treated animals. Assuming a comparable degree of isotope re-utilization in the two groups, this approach provides a relative measure of microsomal-protein turnover. In control animals, radioactivity of total microsomal protein decreases with time with a half-time of about 3 days. In phenobarbital-treated animals, however, there is a marked stabilization of microsomal protein so that almost no radioactivity is lost over a S-day period. The reduced protein catabolism is observed both in total microsomes and in a purified microsomal protein, NADPH cytochrome c reductase. Thus, repeated administration of phenobarbital to animals evokes an increase in... 

Laughlin, C., and Strohl, W. A., 1976a, Factors regulating cellular DNA synthesis induced by adenovirus infection I. The effects of actinomycin D or G-arrested BHK21 cells abortively-infected by type 12 adenovirus or stimulated by serum. Virology 74 30. 

Structural proteins. These changing patterns of protein synthesis were seen in clearer detail using autoradiography of dried polyacrylamide gels (Honess and Roizman, 1973). Honess and Roizman (1975) also used cycloheximide to confine viral mRNA synthesis to the production of immediate-early (a) mRNA. They found that, after reversal of cycloheximide, host protein synthesis declined but that the decline was less rapid if actinomycin was added at the same time as removing the cycloheximide, suggesting that p-polypeptides were at least partly responsible for the inhibition of cellular protein synthesis. 

Pseudorabies virus apparently causes delayed, but not early, virion-associated shut-off. The inhibition of cellular protein synthesis by PRY is prevented by actinomycin (Ben-Porat et ah, 1971 Ihara et al., 1984). By labeling proteins during the hour immediately following reversal of a cycloheximide block it was shown that host pro-... 

Ihara et al. (1983), working with PRV, found that it differs from HSV-1 in that only one immediate-early polypeptide, with an electrophoretic mobility similar to that of ICP4 of HSV, was made after reversing a cycloheximide block. Cellular protein synthesis was inhibited soon after reversal. is a mutant of PRV with a defect in the immediate-early protein which (like tsK of HSV-1 see Section 7.6) is unable to progress from immediate-early to early protein synthesis at the nonpermissive temperature (41°C). This mutant caused significant shut-off of cellular protein and DNA synthesis at 41°C but less than wild-type virus. It was concluded that the immediate-early protein is involved in the shut-off, but either the mutant form is partially defective in this function or some later viral proteins also contribute to the shut-off by wild-type virus. The case for the host-suppressing function of the immediate-early protein would be strengthened if it were confirmed that shut-off occurred after reversal of cycloheximide in the presence of actinomycin, as was reported for polysome breakdown (Ben-Porat et al., 1971). 

Honess and Roizman (1974) incubated cells infected with HSV-1 in the presence of cycloheximide to allow accumulation of a-mRNA. On removing the inhibitor, viral a-polypeptides as well as cellular polypeptides were made and the rates of synthesis of both declined as (3- and 7-polypeptides were produced. The rates of decline were less if actinomycin was added at the time of removal of cycloheximide, suggesting that (3- or 7-polypeptides were concerned with the inhibition of both host and a-protein synthesis. 

The work of Read and Frenkel (1983) suggests that virion proteins are responsible for both early shut-off of host protein synthesis and shut-off of a-protein synthesis. The mutant vhs (which was defective in the virion-associated host shut-off factor) made a-mRNA that was functionally more stable than that of the parental wild-type HSV-1. If cells infected in the presence of cycloheximide, in which vhs a-mRNA had accumulated, were then treated with actinomycin and superinfected with wild-type HSV-1, the a-polypeptide synthesis was prevented and at the same time host protein synthesis was shut off. It was suggested that the wild-type virus carried in its virion an agent that was responsible for inactivating preformed a-mRNA, or restricting its translation, whereas, vhs was defective in this respect and, hence, that the same virion-associated protein may possibly cause both early host shut-off and the decline of a-polypeptide synthesis. If so, a-mRNA presumably has some structural feature in common with cellular mRNA and different from later viral mRNA and it would not be surprising if the p- or 7-protein that causes delayed shut-off may also (or in some cases, alternatively) be involved in the inactivation of a-mRNA. 

Taylor (1965) showed that the amount of radioactivity incorporated into RNA is not significantly different in SF virus-infected CEF cells and in mock-infected cells early in infection. During the exponential phase of viral replication, however, about 70-90% of the newly synthesized RNA is virus specific, since it can be synthesized in the presence of actinomycin D. Mussgay et al. (1970) demonstrated almost the same inhibition pattern in CEF cells infected with SIN virus. Moreover, infection of BHK cells with WEE virus did not enhance cellular RNA degradation (M. Wagatsuma and B. Simizu, unpublished data). 

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