Inhibition Of Total RNA Synthesis Research Articles

8-Chloro-Adenosine Inhibits Molecular Poor-Risk Acute Myeloid Leukemia (AML) and Leukemic Stem Cells (LSC) Growth and Synergizes with the BCL-2 Inhibitor Venetoclax (ABT-199)

Only a minority of AML patients are cured with currently available chemotherapy regimens. Thus, new drugs with novel mechanisms of action are urgently needed. In previous proof-of-concept studies, the halogenated ATP analog 8-chloro-adenosine (8-Cl-Ado) has shown preclinical activity against a variety of solid tumors and hematologic malignancies, favorable pharmacokinetic and pharmacodynamic profiles and feasibility in a first-in-man phase I clinical trial in chronic lymphocytic leukemia. However, little is known regarding the activity of this compound in AML. In contrast to other nucleoside analogs used for treatment of AML, 8-Cl-Ado is metabolized by adenosine kinase and its triphosphate derivative is incorporated into RNA and significantly inhibited RNA synthesis in AML cells (KG-1a and MV4-11 cell lines and primary bone marrow samples) in a dose-dependent manner (300 nM to 1 uM) after 24 h exposure as compared to vehicle-treated controls (p<0.05). Similarly, 20-80% inhibition of total RNA synthesis was observed in AML patient blasts, regardless of the harbored gene mutations or cytogenetic aberrations. In contrast, 24 h exposure to 8-Cl-Ado did not significantly inhibit DNA synthesis compared to vehicle at all concentrations tested (p>0.25 at all concentrations tested). 8-Cl-Ado was metabolized into its cytotoxic triphosphate, 8-Cl-ATP, which accumulated to intracellular concentrations of more than 600 uM after 12 h exposure to 10 uM 8-Cl-Ado. Accumulation of 8-Cl-ATP was associated with >20% reduction of endogenous ATP compared to control-treated cells (p<0.05). In regard to antileukemia activity, 8-Cl-Ado inhibited growth of several AML cell lines (MOLM-13, MOLM-14, KG-1a, MV4-11, OCI-AML3) with IC50s ranging from 0.2 uM to 1.4 uM after 72 h of treatment and that of primary AML blasts including those harboring the poor-risk FLT3-ITD mutation (IC50: 800 nM for FLT3-ITD-positive blasts). In an orthotopic mouse model where FLT3-ITD-positive MOLM-14 AML cells were xenografted, animals treated with 50 mg/kg/day through an implanted osmotic pump had >70% reduction in tumor mass compared to vehicle-treated controls after 16 days of treatment (p<0.05). Importantly, 24 h pre-treatment of human LSC-enriched CD34+CD38- blasts with 5 uM 8-Cl-Ado resulted in a significant inhibition of their colony forming ability compared to vehicle-treated controls (>50% reduction of colonies after 14 days, p<0.05). To study the effect of 8-Cl-Ado on primary AML in vivo, sub-lethally irradiated Rag-2/gamma(c) double-knockout immunodeficient mice were injected retro-orbital with ten million primary human AML blast cells pre-treated with 5 uM 8-Cl-Ado or vehicle control 24 h prior to i.v. injection. Significant longer survival was observed of mice engrafted with drug-treated cells compared to those engrafted with vehicle-treated controls (p<0.0004). Given the previously reported higher expression of the antiapoptotic BCL-2 protein and the implication that this had on treatment resistance in AML, combination strategy using 8-Cl-Ado together with the BCL-2 inhibitor venetoclax was investigated and showed strong synergistic, anti-proliferative effects in both AML cell lines and primary AML blasts carrying a variety of mutations and cytogenetic aberrations. Combination index value calculation and isobologram display demonstrated strong synergy between venetoclax and 8-Cl-Ado in all 4 AML cell lines and 2 primary AML samples investigated, at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90). We also show that, in KG-1a and MV4-11 cells, 8-Cl-Ado and venetoclax synergize in the inhibition of intracellular ATP pools. AML cell lines were treated for 6h with 10 uM 8-Cl-Ado, 250 nM venetoclax or both, before measurement of intracellular ATP. While single agent treatment decreased intracellular ATP levels in both cell lines by about 20% (8-Cl-Ado) to 40% (venetoclax), combination treatment lowered ATP levels by about 70% (KG-1a) to 90% (MV4-11). Taken together, 8-Cl-Ado is a promising agent with a unique RNA and ATP-targeting mechanism of action, excellent toxicity profile and encouraging preclinical anti-leukemia activity in AML and synergizes with the BCL-2 inhibitor venetoclax in vitro. Based on these results, we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory AML and planned further combination with BCL-2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

8-Chloro-Adenosine Inhibits Molecular Poor-Risk Acute Myeloid Leukemia (AML) and Leukemic Stem Cells (LSC) Growth Via Novel RNA- and ATP-Directed Mechanisms: A Novel Therapeutic Approach for AML

Only a minority of patients with AML are cured with currently available chemotherapy regimens. Thus, new drugs with novel mechanisms of action are urgently needed. In previous proof-of-concept studies, the halogenated ATP analog 8-chloro-adenosine (8-Cl-Ado) has shown activity against a variety of solid tumors and hematologic malignancies in vitro, favorable pharmacokinetic and pharmacodynamic profiles in preclinical animal studies and feasibility in a first-in-man phase I clinical trial in chronic lymphocytic leukemia (CLL), where plasma concentrations up to 1 uM could be achieved; since maximum tolerated dose was not yet reached in the CLL clinical trial, higher plasma concentration of 8-Cl-Ado can be anticipated. However, little is known regarding the activity of this compound in AML. In contrast to other nucleoside analogs used for treatment of AML (e.g., cytarabine, azanucleosides), 8-Cl-Ado is metabolized by adenosine kinase and its triphosphate derivative is incorporated into RNA and significantly inhibited RNA synthesis in AML cells (KG1a and MV-4-11) in a dose dependent manner (300 nM to 1 uM) after 24 h exposure as compared to vehicle-treated controls (p<0.05 at 300 nM 8-Cl-Ado). Similarly, 20-80% inhibition of total RNA synthesis was observed in AML patient blasts, regardless of the harbored mutational or cytogenetic aberrations. This resulted in changes in expression levels of both coding and non-coding RNA (e.g. miR-126) in immunophenotypically distinct AML cell populations including leukemia-stem cell (LSC)-enriched CD34+CD38-fractions. In contrast, 24 h exposure to 8-Cl-Ado did not significantly inhibit DNA synthesis compared to vehicle at any concentration tested (p>0.25 at all concentrations tested). 8-Cl-Ado was metabolized into its cytotoxic triphosphate, 8-Cl-ATP, which accumulated to intracellular concentrations of more than 600 uM after 12 h exposure to 10 uM 8-Cl-Ado. Accumulation of 8-Cl-ATP was associated with >20% reduction of endogenous ATP compared to control-treated cells (p<0.05). In regard to antileukemia activity, 8-Cl-Ado inhibited growth of AML cell lines (i.e., Molm-13, Molm-14, KG1a, MV-4-11, OCI-AML3) with IC50s ranging from 0.2 uM to 1.4 uM after 72 h of treatment. 8-Cl-Ado was also active against primary AML blasts including those harboring the poor-risk FLT3 -ITD mutation (IC50: 800 nM for FLT3-ITD-positive blasts). Furthermore, the effect of 8-Cl-Ado on tumor cells has been demonstrated to not be affected by p53 status. It is important to point out that loss of p53 and mutations in p53 are associated with drug resistance and adverse outcomes in AML. In an orthotopic mouse model where FLT3 -ITD-positive Molm-14 AML cells were xenografted, animals treated with 50 mg/kg/day through an implanted osmotic pump had >70% reduction in tumor mass compared to vehicle-treated controls after 16 days of treatment (p<0.05). Importantly, 24 h pre-treatment of human LSC-enriched CD34+CD38- blasts with 10 uM 8-Cl-Ado resulted in a significant inhibition of their colony forming ability compared to vehicle-treated controls (>50% reduction of colonies after 14 days, p<0.05). To study the effect of 8-Cl-Ado on AML LSCs in vivo, sub-lethally irradiated Rag-2/gamma(c) double-knockout immunodeficient mice were i.v.-injected with ten million human LSC-enriched CD34+CD38- blasts pre-treated with 10 uM 8-Cl-Ado or vehicle control 24 h prior to i.v. injection. Significant longer survival was observed of mice engrafted with drug-treated cells compared to those engrafted with vehicle-treated controls (p<0.0004).Taken together, 8-Cl-Ado is a promising agent with a unique RNA and ATP-targeting mechanism of action, excellent toxicity profile and encouraging preclinical anti-leukemia activity in AML. Based on these results, we have initiated a phase I/II clinical trial in patients with relapsed/refractory AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 343: Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia

Abstract Recently, deep sequencing technology identified SF3B1, a gene encoding a splicing factor, is recurrently mutated in chronic lymphocytic leukemia (CLL). Moreover, its mutation was associated with rapid disease progression and resistance to treatment. SF3B1 is a critical component of the spliceosome, leading to the potential of splicing regulation in CLL pathogenesis. However, the mechanism by which SF3B1 mutations affect CLL disease progression is not known. CLL cells are characterized by their resistance to apoptosis, driven mostly by the overexpression of the Bcl-2 family anti-apoptotic proteins. Many members of the family have multiple splicing variants, sometimes encoding proteins with totally opposite functions. For example, the alternatively spliced, shorter variant of the anti-apoptotic protein Mcl-1, Mcl-1S, resembles BH3-only proteins and induces apoptosis. Using herboxidiene, an inhibitor of SF3B1, we explored the activity of SF3B1 in splicing control of Mcl-1 in primary CLL cells. Primers and probes were designed to specifically detect the Mcl-1 pre-mRNA, the anti-apoptotic Mcl-1 and pro-apoptotic Mcl-1S by real-time RT-PCR. We found that inhibition of SF3B1 reduced the level of Mcl-1 in a concentration dependent manner, which was accompanied by the simultaneous surge of Mcl-1S mRNA by over 10 fold. Immunoblots confirmed reduction of Mcl-1L and concurrent increase of Mcl-1S protein. These results indicated a major role of SF3B1 in Mcl-1 splicing control toward the anti-apoptotic variant. In contrast, although also depleting the Mcl-1 mRNA, the Cdk9 inhibitor SNS-032 reduced both the long and short forms of Mcl-1, as well as its pre-mRNA, consistent with its inhibition on transcription. Uridine incorporation measurements showed inhibition of total RNA synthesis by herboxidiene at concentrations above 30 nM, but dramatic reversion of Mcl-1 splicing was observed at 10 nM, which did not affect RNA synthesis, suggesting that the major mechanism of herboxidiene in CLL cells was modification of Mcl-1 splicing. As CLL cells are critically dependent on Mcl-1 for survival, reducing its level induced mitochondrial membrane permeabilization and apoptosis, which was further accelerated by the accumulation of Mcl-1S. Apoptosis occurred in a time- and concentration- dependent manner, with IC50 of less than 100 nM after 24 hr of incubation. Herboxidiene toxicity was selective to CLL B cells, while sparing the normal lymphocytes from healthy donors. Herboxidiene was equally toxic to CLL cells harboring SF3B1 mutations compared to cells carrying the wild-type SF3B1 (n=5 each). Real-time RT-PCR showed similar increase in the ratio of short to long forms of Mcl-1 by herboxidiene in these two groups. Together, these studies identified SF3B1 mediated splicing control as a novel target for CLL therapy. Further investigations are needed to understand the mechanism of SF3B1 mutations on the control of mRNA splicing. Citation Format: Rong Chen, Yuling Chen, Emanuela M. Ghia, Laura Rassenti, Thomas Kipps, Michael J. Keating, William G. Wierda, William Plunkett. Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2014-343

6-(Het)aryl-7-Deazapurine Ribonucleosides as Novel Potent Cytostatic Agents

A series of novel 7-deazapurine ribonucleosides bearing an alkyl, aryl, or hetaryl group in position 6 and H, F, or Cl atom in position 7 has been prepared either by Pd-catalyzed cross-coupling reactions of the corresponding protected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with alkyl- or (het)arylorganometallics followed by deprotection, or by single-step aqueous phase cross-coupling reactions of unprotected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with (het)arylboronic acids. Significant cytostatic effect was detected with a substantial proportion of the prepared compounds. The most potent were 7-H or 7-F derivatives of 6-furyl- or 6-thienyl-7-deazapurines displaying cytostatic activity in multiple cancer cell lines with a geometric mean of 50% growth inhibition concentration ranging from 16 to 96 nM, a potency comparable to or better than that of the nucleoside analogue clofarabine. Intracellular phosphorylation to mono- and triphosphates and the inhibition of total RNA synthesis was demonstrated in preliminary study of metabolism and mechanism of action studies.

Oxidized low-density lipoproteins inhibit endothelial cell proliferation by suppressing basic fibroblast growth factor expression.

Hyperlipidemia inhibits proliferation of endothelial cells (ECs) in culture and angiogenesis in vivo and in arterial explants. Elucidation of the mechanisms may suggest novel therapies against atherosclerosis. Basic fibroblast growth factor (bFGF) expression and mitogenic effects were assessed in bovine aortic ECs incubated with oxidized LDL (ox-LDL). Compared with native LDL and lipoprotein-free controls, ox-LDL reduced bFGF mRNA levels in a time- and concentration-dependent manner, 100 microg/mL producing a maximum reduction of 40% to 50% within 24 to 48 hours. There were commensurate reductions in intracellular and extracellular bFGF concentrations, DNA and total RNA syntheses, and cell replication. FGF receptor 1 and beta-actin mRNA levels were unchanged. Ox-LDL accelerated bFGF mRNA degradation in actinomycin D-treated cells. However, inhibition of bFGF expression by ox-LDL was attenuated by cyclohexamide, indicating a requirement for continuous new protein synthesis for posttranscriptional destabilization. Reduced syntheses of DNA and total RNA were completely restored by bFGF but not by vascular endothelial growth factor. Inhibition of total RNA synthesis achieved by exposing cells to a bFGF-neutralizing antibody was similar in magnitude to that induced by ox-LDL. Cytotoxic effects of ox-LDL on ECs are attributable in part to suppression of bFGF expression.

Effect of DNA-interacting drugs on phage T7 RNA polymerase.

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

Cobalt exerts opposite effects on erythropoietin gene expression in rat hepatocytes in vivo and in vitro.

This study investigates the effects of hypoxia and of cobalt on erythropoietin (EPO) gene expression in hepatocytes in vivo and in vitro in neonatal, juvenile, and adult rats. With the use of the ribonuclease protection assay to quantify RNA, both hypoxia (0.1% CO or 9% O2) and cobalt (60 mg/kg) elicit production of increased amounts of EPO mRNA in neonatal and juvenile rat liver in vivo. In vitro hepatocyte EPO gene expression could be reproducibly stimulated by hypoxia (3% O2) but not by cobaltous chloride (50-150 microM) within 2-20 h. Conversely, cobalt substantially attenuated the rise of EPO mRNA levels in response to hypoxia. This inhibitory effect of cobalt was mimicked by zinc but not by other metals. CO attenuated the rise of EPO mRNA levels in vitro in response to hypoxia; this inhibitory effect coincided with an inhibition of total RNA synthesis as determined by [3H]uridine incorporation. The lack of specific inhibitory effects of CO and of specific stimulatory effects of cobalt on hepatocyte EPO gene expression in vitro suggests that a specific heme oxygen sensor may be less important than in hepatoma cells.

Putative determinants of the cellular response to hyperthermia.

Recently, it has been demonstrated that two different thermal resistant states found in Chinese hamster cells, one transient, associated with thermotolerance, and the other permanent, associated with the increased expression of the cognate member of the hsp 70 family, are characterized by faster recovery from heat-induced perturbations in several cellular processes (Laszlo 1992b). These processes include total cellular protein and RNA synthesis, the localization of hsp70, the organization of vimentin, and the protein composition of the nucleus. In the present study, the recovery from heat-induced perturbations in cellular physiology was extended further to two more types of Chinese hamster cells: permanently heat resistant cells in which thermoresistance is associated with the overexpression of hsp27 and heat-sensitive cell lines. When the heat-resistant hsp27 transfected cell lines were compared with the control wild-type cell line, the recovery of protein synthesis from heat-induced inhibition was similar in the normal and hsp27 transfected cells, while the recovery from heat-induced inhibition of total RNA synthesis and the recovery from heat-induced increased association of hsp70 with nuclei were both more rapid in the hsp27 transfected cell lines. In the permanently heat-sensitive cell lines, the kinetics of recovery from heat-induced inhibition of protein synthesis did not correlate with the heat sensitive state. However, delays in the recovery from heat-induced alterations in total cellular RNA synthesis and from heat-induced excess nuclear association of hsp70 were associated with the heat-sensitive state. Overall, these results suggest that the kinetics of recovery from heat-induced alterations in total cellular RNA synthesis and the localization of hsp 70 are putative candidates for being determinants of the cellular response to hyperthermia, and thus have the potential to form the basis of predictive assays for use in conjunction with clinical hyperthermia.

The role of guanosine-3',5'-bis-pyrophosphate in mediating antimicrobial activity of the antibiotic 3,5-dihydroxy-4-ethyl-trans-stilbene.

The mode of action of 3,5-dihydroxy-4-ethyl-trans-stilbene (ES), an antibiotic produced by Xenorhabdus luminescens symbiotically associated with an entomopathogenic nematode, was investigated. ES was active against gram-positive and a number of gram-negative bacteria. In susceptible bacteria this antibiotic caused the inhibition of total RNA synthesis and, to a lesser extent, protein synthesis. At or above MICs, ES triggered a substantial accumulation of an intracellular regulatory compound, guanosine-3',5'-bis-pyrophosphate (ppGpp). This response was also noticed in species of bacteria which have previously not been shown to use ppGpp as a regulatory molecule. The involvement of ppGpp in antibiotic action was confirmed by using an isogenic stringent and a relaxed pair of Escherichia coli strains. The fact that the accumulation of ppGpp was correlated with the susceptibility of various gram-positive and gram-negative bacteria to ES suggests that this nucleotide is involved in the regulation of RNA synthesis and growth in all these microorganisms. Thus, inhibition of RNA synthesis via an increase in ppGpp concentrations may represent a mechanism that is prevalent among most bacteria and one that could be exploited for achieving a rapid inhibition of bacterial growth.

RNA synthesis during germination of UV-irradiated Dictyostelium discoideum spores.

UV irradiation to the spores of Dictyostelium discoideum NC4 resulted in a more prolonged delay of amoeba-emergence from swollen spores with increasing UV fluence. During the germination, an inhibition of total RNA synthesis and a shift of stage of maximum RNA synthesis to the later period were observed. The maximum poly(A)+ RNA synthetic activity was found on an early stage of amoeba-emergence prior about 1 h to the beginning of rRNA synthesis in unirradiated spore germination; but, in UV-irradiated spore germination, the stage of maximum poly(A)+ RNA synthesis shifted to the later stage of germination with increasing UV fluence. A decreased synthesis of poly(A)+ RNA and a severe inhibition of rRNA synthesis were observed on UV-irradiated and germinated spores, but no significant inhibition of 4-5 S RNA synthesis was detected. Actinomycin D suppressed almost completely the rRNA synthesis of emerged amoebae but the drug apparently did not affect the emergence of amoebae at any stage of germination. It was postulated that the delay of amoeba-emergence in UV-irradiated spore must be mainly due to the shift of the stage of maximum synthesis of poly(A)+ RNA to the later stage of germination.

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.

Actinomycin D in Tetrahymena: Non-specific inhibition of RNA synthesis and primary and secondary effects on protein synthesis

The effects of actinomycin D on RNA and protein synthesis have been investigated in Tetrahymena pyriformis. The extent of inhibition of total RNA synthesis and the time before inhibition occurs are both dependent on the concentration of the drug over the range 0.5–10 μg/ml. In contrast to reports on mammalian systems, actinomycin D at low doses is not a selective inhibitor of rRNA synthesis in Tetrahymena; at all doses tested, poly(A+) RNA and poly(A−) RNA syntheses were reduced to similar extents. Finally, the action of actinomycin D was studied in conjunction with γ-radiation which alone causes a transient disaggregation of the polysomes. Actinomycin D partially inhibits the reassociation of polysomes in irradiated cells, but after recovery, the polysome content of the irradiated and drug-treated cells is greater than that of cells treated only with actinomycin D. The results support both a direct action of actinomycin D on translation and secondary effects resulting from inhibition of transcription. The effect of actinomycin D on protein synthesis was determined by two independent methods. 1. 1. Investigation of drug-induced alterations in polysome content showed that as the dose increases ( a) the lag period before observable polysome dissociation decreases; ( b) the rate of polysome dissociation increases; and ( c) the final extent of polysome loss increases. 2. 2. By measuring the relative rates of incorporation of [ 35S]methionine into N-terminal and internal positions of nascent peptides, it was determined that actinomycin D inhibits initiation and elongation to similar extents.

Hormonal control of hyaluronic acid production in fibroblasts and its relation to nucleic acid and protein synthesis

Whole serum and elevated pH previously had been found to stimulate both cell multiplication and hyaluronic acid production by chick embryo fibroblasts in culture. In a study to determine whether cell multiplication and hyaluronic acid production both respond to a single well-defined substance, insulin was found to stimulate, and cortisol to inhibit both processes coordinately. It appears, therefore, that multiplication and differentiated function in fibroblasts respond to a common underlying regulatory signal. Inhibition of ribosomal RNA synthesis by actinomycin D does not prevent serum stimulation of hyaluronic acid production, but inhibition of total RNA synthesis does. If total RNA synthesis is inhibited only after the hyaluronic acid production has reached a new high level, it continues at that level for the next five hours. The stimulatory treatment causes an increase in the activity of the enzyme hyaluronate synthetase. Inhibition of protein synthesis prevents any increase in hyaluronic acid production, and reduces the basal level of production. Reduction of the availability of Mg2+ in the medium coordinately inhibits DNA synthesis and hyaluronic acid production. The results are discussed in the light of a model for coordinate control growth and metabolism based on the availability of Mg2+.

Suppression of interferon production by vitamin A.

Vitamin A (retinoic acid) suppressed interferon production by L cells infected with Newcastle disease virus (NDV). This suppression was maximal when cells were treated with retinoic acid for 2 h after NDV adsorption, indicating that the inhibitory step was an early event. It was not due to inhibition of total RNA synthesis. Retinoic acid treatment caused both a delay in appearance of interferon and a reduced rate of synthesis thereafter.

THE INHIBITION OF FUNCTIONAL RNA SYNTHESIS IN CHLORELLA PYRENOIDOSA BY 6‐METHYL PURINE

SummaryAddition of 10‐3 M 6‐methyl purine caused immediate inhibition of total RNA synthesis in Chlorella pyrenoidosa, 211‐8p, whereas inhibition of total protein synthesis developed progressively with time. 50% inhibition of adenine incorporated into RNA, and of de novo isocitrate lyase synthesis, was caused by 5 × 10‐5 M 6‐methyl purine, but a ten‐fold higher concentration was required to give a similar inhibition of total RNA synthesis. The inhibition of isocitrate lyase synthesis was reversed competitively by adenine. Isocitrate lyase synthesis was inhibited by 91% at a concentration of 10‐3 M 6‐methyl purine, which gave only 12% inhibition of glucose respiration. It is suggested that 6‐methyl purine acts by replacing adenine in RNA metabolism. It is concluded that 6‐methyl purine may be used as an inhibitor of functional RNA synthesis in Chlorella and that the synthesis of isocitrate lyase during the cell cycle is dependent upon concurrent transcription.

Effects of abscisic acid on nucleic acid metabolism in maize coleoptiles

Following treatment with ABA an inhibition of total RNA synthesis was observed after 30 hours. Total soluble ribonuclease activity did not change during the first 8 hours, after which an increase could be observed.Separation of nucleic acids with polyacrylamide gel electrophoresis indicated that synthesis of soluble RNA was less inhibited by ABA than synthesis of ribosomal RNA.Effects of 5-FU and ABA on ribosomal RNA precursor were investigated. It could be shown that 5-FU did not inhibit ribosomal precursor synthesis, but that ABA did so.

Effects of X-Irradiation on RNA and Protein Synthesis in HeLa Cells

Alterations in protein and RNA metabolism in asynchronously growing HeLa cell population following 2000 rads of x-irradiation were studied using sucrose density gradients, radioactive precursor labeling, and autoradiographic procedures. It was found that irradiation reduced protein synthesis within a 2-hour period while a depression of RNA synthesis by x-irradiation was evident somewhat later. Rapidly labeled ribosome precursor RNA was delayed in its transformation into smaller RNA molecules as revealed by pulse chase experiments at a time when inhibition of total RNA synthesis becomes noticeable after 2000 rads of x-irradiation. Specific activities of polysomal fractions from irradiated and control cells labeled with radioactive amino acids are almost identical, but the total amount of radioactivity was less in the sedimentation profile of irradiated cells than in controls. Several possible mechanisms that could result in the depression of protein synthesis are discussed.

INHIBITION OF SYNCHRONIZED CELL DIVISION IN TETRAHYMENA BY ACTINOMYCIN D.

The effects of actinomycin D on RNA and protein synthesis have been investigated in Tetrahymena pyriformis. The extent of inhibition of total RNA synthesis and the time before inhibition occurs are both dependent on the concentration of the drug over the range 0.5–10 μg/ml. In contrast to reports on mammalian systems, actinomycin D at low doses is not a selective inhibitor of rRNA synthesis in Tetrahymena; at all doses tested, poly(A+) RNA and poly(A−) RNA syntheses were reduced to similar extents.Finally, the action of actinomycin D was studied in conjunction with γ-radiation which alone causes a transient disaggregation of the polysomes. Actinomycin D partially inhibits the reassociation of polysomes in irradiated cells, but after recovery, the polysome content of the irradiated and drug-treated cells is greater than that of cells treated only with actinomycin D. The results support both a direct action of actinomycin D on translation and secondary effects resulting from inhibition of transcription.The effect of actinomycin D on protein synthesis was determined by two independent methods. 1.1. Investigation of drug-induced alterations in polysome content showed that as the dose increases (a) the lag period before observable polysome dissociation decreases; (b) the rate of polysome dissociation increases; and (c) the final extent of polysome loss increases.2.2. By measuring the relative rates of incorporation of [35S]methionine into N-terminal and internal positions of nascent peptides, it was determined that actinomycin D inhibits initiation and elongation to similar extents.