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The production of cytoplasmic RNA that contains polyadenylic acid is increased, relative to total cytoplasmic RNA, in a neuroblastoma clone, NBE-(A), after induction of differentiation by 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, an inhibitor of adenosine 3',5'-monophosphate phosphodiesterase. The amount of RNA that contains polyadenylic acid in cytoplasm may be greater in such differentiated neuroblastoma cells than in proliferating control cells.

Mouse myeloma cells have previously been shown (L. B. Schwartz, V. E. F. Sklar, J. A. Jaehning, R. Weinmann & R. G. Roeder, submitted for publication) to contain two chromatographically distinct forms of RNA polymerase III (designated III(A) and III(B)). The enzymes are unaffected by low alpha-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for III(A) and III(B)) at higher toxin concentrations. RNA polymerase I was unaffected at all alpha-amanitin concentrations tested. Myeloma RNA polymerases II, III(A), and III(B) appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic alpha-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases. Isolated myeloma nuclei and nucleoli contińue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by alpha-amanitin, but only at high concentrations; and the alpha-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases III(A) and III(B). In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by alpha-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high alpha-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems. These results unequivocally demonstrate the role of RNA polymerase III (III(A) and/or III(B)) in the synthesis of (pre) 4S RNAs and a 5S RNA species.

In the Reuber (H35) hepatoma cell strain, microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase is induced 25-fold by the polycyclic hydrocarbon benz[a]anthracene but is not induced by the steroid hormone dexamethasone. Soluble tyrosine aminotransferase is induced sixfold by dexamethasone and twofold by benz[a]anthracene. Each enzyme requires similar inducer concentrations for induction, and their induction kinetics are similar. The induction of each enzyme requires RNA and protein synthesis; in each case the transcriptional and translational steps can occur independently. The two induction systems are differentially sensitive to inhibitors of macromolecular synthesis. Simultaneous exposure to both inducers produces increases in both enzyme activities that are greater than those produced by either inducer alone. Each inducer acts at a pretranslational level to produce this synergistic effect. The results suggest that the requirements for macromolecular synthesis are similar for the induction of each enzyme, but that the turnover of enzyme-specific macromolecules may differ for each.

Inhibition by double-stranded polyribonucleotides of DNA synthesis in synchronized HeLa cultures is dose- and time-dependent. Inhibition by poly(I.C) primarily affected late G(1) and early S phases of the cell cycle. Single-stranded polynucleotides, native calf-thymus DNA, and yeast RNA had no effect. Radioautography showed that after 2-hr exposure the synthetic polyribonucleotides were predominantly inside the nucleus. The results extend the spectrum of action of double-stranded RNA.

One possible mechanism of virus-induced cell damage is that the redistributed (released) lysosomal enzymes produce the cytopathic effect during cytolytic types of infections such as poliovirus in HEp-2 cells. To determine if the lysosomal enzyme redistribution and cell damage are host-cell directed, we studied sensitivity of these events to the action of actinomycin D. By the use of actinomycin D at concentrations producing the least toxicity but maximal effectiveness in shuting down cell RNA synthesis, it was shown that the cytopathic effect and enzyme redistribution were not inhibited and, therefore, not directly controlled and induced by the cell genome in response to the virus infection. Evaluation of cytopathic effect by a phase contrast microscopy method detected changes earlier than the erythrocin B uptake method.

The effect of encephalomyocarditis virus infection of MOPC 460 mouse plasmacytoma cells on host RNA synthesis and RNA polymerases was investigated. Consistent with work performed in other virus host systems, rates of RNA synthesis appeared to be inhibited in infected cells, whereas RNA degradation appeared normal. These results were further extended with isolated nuclei, in which distinct RNA polymerase activities could be studied under conditions where problems with RNA turnover and endogenous nucleotide pool sizes were insignificant. Endogenous nuclear RNA polymerase II activity was inhibited early postinfection and at 1 to 2 h prior to endogenous RNA polymerase I plus III activity. However, the solubilized enzymes were fully active with exogenous DNA as template. In fact, the levels of RNA polymerases I, II, and III, isolated from infected cells and nuclei, were indistinguishable from levels in uninfected cells and nuclei at each stage of their partial purification procedure. The chromatographic properties of the enzymes on DEAE-Sephadex were also unaltered. Furthermore, the RNA synthetic activity of these isolated enyzmes, or of nuclei isolated from uninfected cells, was resistant to extracts of nuclei or of cytoplasmic fractions from infected cells. These results are discussed in terms of a possible inhibition of RNA synthesis in vivo at the level of transcription initiation.

Tumor angiogenesis factor (TAF) and its importance in determining a strategy for cancer chemotherapy are discussed. It is suggested that inhibition of RNA synthesis or increased RNA catabolism might interfere with the metabolism of solid tumor cells more so than in normal cells, and thus hinder angiogenesis and pursuant tumor growth by preventing the synthesis of the RNA component of TAF. An attempt is made to indicate potential models for anti-angiogenesis agents of this type. The drugs offered as initial prototypes for investigations along these lines are actinomycin D (which likely has antimetabolite and anti-angiogenesis activities), polyriboinosinic-polyribocytidylic acid (which likely has adjuvant and anti-angiogenesis activities) and ribonuclease (which in theory might be a purely anti-angiogenetic agent). It is noted that these models may turn out to be less than ideal as therapeutic agents due to problems of toxicity, metabolism, potency, or distribution, but nonetheless might serve to yield insights into the design of new cancer chemotherapeutic drugs. In addition, some evidence is cited suggesting that actinomycin D may be more effective against certain tumors when employed in lower, chronic dosages rather than its present use in "loading" dosages.The concept of anti-angiogenesis agents as fundamentally "tumoristatic" therapies is discussed, and the likelihood that such agents might be effectively "tumoricidal" in immunocompetent hosts is mentioned. The main promise of an anti-angiogenetic strategy is efficacy against presently intractable slowly growing human cancers when used in combination with other treatment modalities. In summary, a strategy of cancer chemotherapy predicated upon interference with RNA synthesis or increase in RNA catabolism is offered as a potential mechanism for establishing anti-angiogenesis, and as a promising alternative and adjunct to present methods.

Chinese hamster and mouse sarcoma 180 ascites cells, treated with high levels of actinomycin D, still carry out limited poly(A) synthesis. The residual activity, which consists of poly(A) chain extension in the cytoplasm as well as in the nucleus, is essentially insensitive to cordycepin. Nuclear polyadenylation proceeds linearly and involves the gradual extension of unusually long poly(A) sequences. Cytoplasmic poly(A) labeling is initially more rapid than in the nucleus, but levels off within 5-10 min. It consists of addition of seven to eight AMP residues to the poly(A) sequences in preexisting mRNA molecules. The levelling off can be accounted for by a rapid turnover of the extremity of the poly(A) chain in the mRNA. Cytoplasmic poly(A) chain extension can be detected in cells not subjected to the actinomycin treatment. The rate of this process is of the same order of magnitude as that of new poly(A) transfer from nucleus to cytoplasm. It could serve to control the length of the poly(A) sequence in mRNA.