Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein SufI can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.

Transient ribosomal attenuation coordinates protein synthesis and co-translational folding

The mechanism of hepatocarcinogenesis caused by peroxisome proliferators (PP) is poorly understood, making it difficult to predict the carcinogenicity of PP to rodents or other species. It has been suggested that the carcinogenic potential of individual PP in rodents is correlated with the degree of PP-induced hepatic peroxisome proliferation. To evaluate this possible correlation, di(2-ethylhexyl)phthalate (DEHP) at 1.2% and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) at 0.1% were fed to male F-344 rats for up to 365 days and hepatocytic peroxisome proliferation and DNA replication were measured. All rats fed Wy-14,643 for 365 days had numerous grossly visible nodules in comparison to none in the livers of DEHP-fed or control rats. Despite this difference in the induction of tumors, both DEHP and Wy-14,643 increased the peroxisomal volume density 4- to 6-fold from 8 to 365 days of treatment. Peroxisomal beta-oxidation enzyme activities were increased 8-fold by both DEHP and Wy-14,643 after 18 days. At later time points (77 to 365 days), these enzyme activities were about 25% higher in livers of Wy-14,643- than DEHP-fed rats. DEHP or Wy-14,643 increased absolute liver weights 50 to 75% above controls after 18 to 365 days of feeding. Labeling of hepatocyte nuclei with a single injection of tritiated thymidine revealed a rapid burst in replicative DNA synthesis in both DEHP and Wy-14,643-fed rats, with a return to control levels by 4 days. Additional rats were implanted with 7-day osmotic pumps containing tritiated thymidine. With this more extended method of labeling a 5- to 10-fold increase in replicative DNA synthesis was observed in rats receiving Wy-14,643 for 39 to 365 days as compared to DEHP-fed rats or controls. In conclusion, when performed under conditions similar to the tumorigenicity studies, the degree of peroxisome proliferation correlated poorly with the relative hepatocarcinogenicity of DEHP and Wy-14,643. However, a strong correlation was observed between the relative hepatocarcinogenicity of DEHP and Wy-14,643 and the ability to induce a persistent increase in replicative DNA synthesis. These data emphasize the possible importance of cell replication in the mechanism of PP-induced hepatocarcinogenesis.

/pdf/relationship-of-hepatic-peroxisome-proliferation-and-403pv0y7ee.pdf

Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) in rats.

Increases in cell proliferation are widely viewed as being of importance in carcinogenesis. We report that exposure of normal human lung fibroblasts to a low dose of α particles like those emitted by radon/radon progeny stimulates their proliferation in vitro , and this response also occurs when unirradiated cells are treated with supernatants from α-irradiated cells. We attribute the promitogenic response to superoxide dismutase- and catalase-inhibitable α particle-induced increases in the concentrations of transforming growth factor β1 (TGF-β1) in cell supernatants. TGF-β1 at concentrations commensurate with those in the supernatants capably induces increases in intracellular reactive oxygen species (ROS) in unirradiated cells. Furthermore, the addition of supernatants from α-irradiated cells to unirradiated cells decreases cellular levels of TP53 and CDKN1A and increases CDC2 and proliferating cell nuclear antigen in the latter. Like the increased intracellular ROS bystander effect, this “decreased TP53/CDKN1A response” can be mimicked in otherwise untreated cells by the addition of low concentrations of TGF-β1. Our results indicate that α particle-associated increases in cell growth correlate with intracellular increases in ROS along with decreases in TP53 and CDKN1A, and that these cellular responses are mechanistically coupled. As well, the proliferating cell nuclear antigen and CDC2 increases that occur along with the decreased TP53/CDKN1A bystander effect also would expectedly favor enhanced cell growth. Such processes may account for cell hyperplastic responses in the conducting airways of the lower respiratory track that occur after inhalation exposure to radon/radon progeny, as well as, perhaps, other ROS-associated environmental stresses.

https://cancerres.aacrjournals.org/content/canres/60/5/1290.full.pdf

Factors underlying the cell growth-related bystander responses to alpha particles.

The sequential analysis of liver cancer induction

Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) are compounds whose physical/chemical properties led to their widespread commercial use. Although their production has been banned or severely limited in most countries since the 1970s, the persistence and stability of these compounds have resulted in a worldwide distribution, especially of PCBs. PBB contamination is limited principally to the state of Michigan, where a series of tragic errors eventually resulted in the accumulation of residues in livestock and the general human population. Long-term exposure to PCBs and PBBs in animals has been associated with the induction of neoplastic nodules in the liver and in some cases hepatocellular carcinoma. This review discusses the role of PCBs and PBBs in the process of carcinogenesis. The mutagenicity/genotoxicity of these compounds, as well as their initiation/promotion potential is discussed. The epidemiology of PCB and PBB exposure is reported along with an estimation of the risk of cancer to humans. Finally, possible molecular mechanisms of action are suggested for polyhalogenated biphenyls in cancer development.

Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs.

CELL proliferation has often been implicated in the development of cancer with chemicals1–3. Supportive evidence for this is the observation that several carcinogens that normally do not induce liver cancer in intact adult animals, especially with a single dose, become carcinogenic if administered as a single injection after partial hepatectomy (PH)1. In these conditions, it is thought that PH may act during initiation, presumably by fixation of some carcinogen-induced DNA damage through replication of the altered DNA. However, because carcinogenesis is a multi-step process, often involving the appearance of several new cell populations between the initial target cells and the ultimate cancer4,5, it is possible that the regenerative response of liver following PH could have a major effect on one or more steps subsequent to initiation. Thus, the use of a very late end-point (cancer) makes it difficult, if not impossible, to relate with any accuracy some very early event to any specific step in the carcinogenic process. Solt and Farber6 have recently developed a new approach to the sequential analysis of carcinogenesis in vivo that delineates the first few steps in the process and is a quantitative assay for initiation of liver cancer. We have used this model to investigate whether cell replication exerts its first effect on initiation or on some later step in the carcinogenic process. And if initiation is at least one site of action, when in the regenerative cell cycle is a carcinogen most effective and what biochemical events might be involved at this phase of the cell cycle?

Initiation of chemical carcinogenesis requires cell proliferation

Adult male Fischer rats were exposed to a necrogenic dose (200 mg/kg) of diethylnitrosamine or to nonnecrogenic doses of N-methyl-N-nitrosourea, 1,2-dimethylhydrazine, or benzo(a)pyrene following partial hepatectomy or sham hepatectomy. This treatment by itself led to no hepatocellular carcinomas by 8 to 18 months, except in animals given N-methyl-N-nitrosourea, which showed a 30% incidence by 12 months. With each treatment regimen, exposure to dietary 2-acetylaminofluorene for 2 weeks coupled with partial hepatectomy or the administration of a necrogenic dose of CCl4, was associated with an incidence of 68 to 94% of cancer at 8, 12, or 18 months, depending upon the initiating carcinogen used. Appropriate controls showed either no hepatocellular carcinoma or a much lower incidence. It is concluded that the 2-week exposure to dietary 2-acetylaminofluorene plus partial hepatectomy or the administration of CCl4 has a strong promoting effect on liver carcinogenesis with four different chemical carcinogens.

https://cancerres.aacrjournals.org/content/canres/43/1/188.full.pdf

Promotion of liver cancer development by brief exposure to dietary 2-acetylaminofluorene plus partial hepatectomy or carbon tetrachloride.

The administration of either N -nitrosodiethylamine (diethyl-nitrosamine) to intact male F-344 rats or N -methyl- N -nitrosourea to similar animals 18 hr after partial hepatectomy results in the induction of altered resistant hepatocytes that persist for up to 36 weeks with no perceptible decrease in their number. The criterion for resistance was the ability to proliferate rapidly and to develop into foci or nodules when exposed to a level of dietary 2-acetylaminofluorene that inhibits the proliferation of the vast majority of hepatocytes when liver cell proliferation is stimulated by surgical or chemical partial hepatectomy. Since this selection procedure, when coupled with a single dose of diethylnitrosamine, is associated with a high incidence of liver cancer as compared to appropriate controls, and since similar foci and nodules were shown previously to be one site of origin for hepatocellular carcinoma in this model, the induction of resistant hepatocytes is interpreted as initiation. Thus, these results suggest that initiation of liver carcinogenesis in this model is irreversible, at least for a period of 36 weeks.

Persistence of Resistant Putative Preneoplastic Hepatocytes Induced by N-Nitrosodiethylamine or N-Methyl-N-nitrosourea

A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.

Edmundo Cayama

Papers

Initiation of chemical carcinogenesis requires cell proliferation

Promotion of liver cancer development by brief exposure to dietary 2-acetylaminofluorene plus partial hepatectomy or carbon tetrachloride.

Persistence of Resistant Putative Preneoplastic Hepatocytes Induced by N-Nitrosodiethylamine or N-Methyl-N-nitrosourea

New chromatographic and biochemical strategies for quick preparative isolation of tRNA

Trypanosoma cruzi: polypeptide markers of epimastigotes and trypomastigotes.