David P. Ringer

Use of terbium fluorescence enhancement as a new probe for assessing the single-strand content of DNA

Abstract Since a previous study showed that the guanosine monophosphate moieties resident in RNA gave markedly greater enhancement of terbium (III) (Tb 3+ ) fluorescence than those resident in an equal amount of DNA, we investigated the possibility that only the guanosine monophosphate moieties residing in single-strand regions of nucleic acids participated in the enhancement of Tb 3+ fluorescence. When native versus thermally denatured DNAs from rat liver were compared, we observed that thermal denaturation of DNA increased its ability to enhance Tb 3+ fluorescence by more than 10-fold. Furthermore, over a range of increasing temperatures (therefore increased thermal denaturation) a positive correlation was demonstrated for increases in Tb 3+ fluorescence enhancement and susceptibility to hydrolysis by the single strand-specific S 1 nuclease. Comparison of changes in Tb 3+ and ethidium bromide fluorescence for native versus thermally denatured DNA indicated that Tb 3+ was five times more sensitive in measuring increases in single-strand content. It is concluded that only those dGuo-5′-P moieties in single-strand DNA are capable of enhancing Tb 3+ fluorescence and, therefore, Tb 3+ fluorescence enhancement can be used to assess the single-strand content of DNA. Standard conditions are described for the use of Tb 3+ fluorescence in the measurement of DNA single strandedness.

Nuclear matrix associated poly(ADP-ribose) metabolism in regenerating rat liver

We have examined a possible role for protein poly(ADP‐ribosylation) during in vivo DNA replication by studying the metabolism of poly(ADP‐ribose) in the nuclear matrix fraction from normal and regenerating rat liver. This fraction contains the newly replicated DNA and thus allows for the examination of the events closely associated with the replication process. It was found that 55% of the total nuclear protein‐bound poly(ADP‐ribose) and 15–35% of the total nuclear poly(ADP‐ribose)‐polymerase activity were tightly associated with this subnuclear compartment in normal liver. Surgical removal of two‐thirds of the liver initiated a time‐dependent decrease in nuclear matrix associated polymers of ADP‐ribose and poly(ADP‐ribose) polymerase activity which reached a minimum of 40% of control livers after 24 h, before returning to normal levels at 41 h post‐partial hepatectomy. In contrast, the total levels of poly(ADP‐ribose) in intact liver and the total polymerase activity of isolated nuclei exhibited a 2‐fold increase over basal levels. These results are consistent with the conclusion that the nuclear matrix is a major poly(ADP‐ribosylation) site within the nucleus and that this metabolic reaction may be closely connected with the events modulating DNA replication in this fraction.

Separation and quantitative analysis of O-linked phosphoamino acids by isocratic high-performance liquid chromatography of the 9-fluorenylmethyl chloroformate derivatives

Phosphoamino acids derivatized with 9-fluorenylmethyl chloroformate were separated on an anion-exchange column (Partisil 10 SAX) at pH 3.90 using an isocratic elution with 10.0 mM potassium phosphate, 1.0% tetrahydrofuran, and 55% methanol. Phosphoamino acids were eluted with baseline resolution in the following order: phosphotyrosine, phosphothreonine, and phosphoserine. Each phosphoamino acid was separated from its parent amino acid, dicarboxylic amino acids, sugaramine phosphates, as well as the other common amino acids. The turn-around time from injection to injection was 35 min. The linearity for all three O-linked phosphoamino acids extended from 0.5-1000 pmol and has been shown to be directly applicable to the analysis of isolated phosphoproteins.

Quantitative analysis of changes in cell proliferation and apoptosis during preneoplastic and neoplastic stages of hepatocarcinogenesis in rat

In situ markers for quantitative analysis of cell proliferation and apoptotic cell death have been used to evaluate both the proliferation level and net growth potential of preneoplastic nodules and malignant tumor tissues from rats experimentally induced for hepatocarcinogenesis by the dietary administration of 2-acetylaminofluorene. The findings show that although tumors have a much higher level of cell proliferation than preneoplastic liver nodules, the nodules have a higher potential for net growth when apoptosis is taken into account. These results support a role for a decrease in apoptosis during the promotion stage of carcinogenesis.

Rapid decrease in N-hydroxy-2-acetylaminofluorene sulfotransferase activity of liver cytosols from rats fed carcinogen

Abstract Sulfation of N -hydroxy-2-acetylaminofluorene ( N -OH-AAF) by N -OH-AAF sulfotransferase yields a candidate for ultimate carcinogen in hepatocarcinogenesis in rats. We have monitored this pathway during the initial phase(s) of hepatocarcinogenesis produced by feeding male Holtzman rats a diet containing 0.05% 2-acetylaminofluorene (AAF). Our studies revealed an immediate and precipitous decrease in N -OH-AAF sulfotransferase activity beginning after 1 day on the AAF diet and decreasing 4- to 5-fold after 5 days on the AAF diet. This decrease in activity remained at low values during continuous administration of AAF throughout 4 weeks but was shown to be both reversible and AAF dose dependent. Parallel monitoring of rat serum glutamic oxaloacetic acid transaminase activity during the administration of AAF indicated that no appreciable hepatocellular toxicity occurred during the period of sulfotransferase activity lowering. Other known carcinogenes, i.e. 3′-methyl- and 4′-fluoro-4-dimethylaminoazobenzene, aflatoxin B 1 , thioacetamide, ethionine, and diethylnitrosamine, and the hepatotoxin α-naphthylisothiocyanate, also caused decreases in N -OH-AAF sulfotransferase activity after 7 and 28 days of administration. In contrast, very weak or non-carcinogens, i.e. p -aminoazobenzene, fluorene, and barbital, failed to reduce N -OH-AAF sulfotransferase activity during 28 days of feeding. Data from these studies on the short-term chronic administration of xenobiotics suggest (a) reduced likelihood for the direct involvement of the sulfotransferase pathway in providing sufficient cytotoxic AAF metabolites to cause compensatory hyperplasia and its putative promotion-effect for AAF-mediated carcinogenesis, and (b) the possible use of the rapid loss in sulfotransferase activity as an early indicator of hepatocarcinogenesis.

Carcinogen-induced alteration in liver epidermal growth factor receptor distribution during the promotion stage of hepatocarcinogenesis in rat.

Changes in hepatic membrane binding capacity for epidermal growth factor (EGF) were assessed during 2-acetylaminofluorine (2-AAF)-induced hepatocarcinogenesis. An overall decrease in membrane EGF binding levels was observed throughout the period of 2-AAF administration. Immunochemical studies indicated the decreases in EGF binding levels were paralleled by decreases in tissue EGF receptor levels. Immunohistochemical studies showed that the losses in hepatic EGF receptor levels were not uniform throughout the liver during the early, promotion stage of cancer development. During the promotion stage, preneoplastic liver nodules were found to display a higher level of EGF receptor than surrounding hepatic tissue. This resistance to 2-AAF-mediated down-regulation of EGF receptor may confer a proliferative advantage to the developing nodule relative to the surrounding liver tissue. Similar immunochemical and immunohistochemical analysis of hepatocarcinomas at late stages of 2-AAF-induced hepatocarcinogenesis indicated a uniform loss of EGF receptor in tumor and surrounding tissue. These studies indicate that carcinogen-mediated changes in EGF binding levels are different during the multistage process of hepatocarcinogenesis, and that resistance to down-regulation of EGF receptor among preneoplastic nodules may have a role in providing a selective growth advantage to initiated cell populations. EGF receptor may be useful as a dynamic marker assessing the development of hepatic tumors.

Sulfation of carcinogenic aromatic hydroxylamines and hydroxamic acids by rat and human sulfotransferases: substrate specificity, developmental aspects and sex differences

Sulfation of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and structurally related hydroxamic acids by rat and human sulfotransferases was studied. There was a clear sex and age difference in the sulfation of N-OH-AAF and the other hydroxamic acids by rat liver cytosols; adult male rats had the highest sulfation activity. Experiments with purified aryl sulfotransferase IV (AST IV) indicated that the high expression of this enzyme in male rat liver may be responsible for these differences. No such sex or age difference was found for the sulfation of aromatic hydroxylamines. In cytosols of adult human livers, sulfation activity towards aromatic hydroxamic acids and hydroxylamines was clearly present, but activities were much lower than in rat liver cytosols. Sulfation activity towards these compounds was also found in fetal and neonatal liver and adrenals. These compounds probably are sulfated by several different sulfotransferases in humans.

Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 +/- 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.

Inhibition of the peptidyl transferase A-site function by 2'-O-aminoacyloligonucleotides.

Abstract New “non-isomerizable” analogs of the 3′-terminus of AA-tRNA, C-A(2′Phe)H, C-A(2′Phe)Me, C-A(2′H)Phe and C-A(2′Me)Phe, were tested as acceptor substrates of ribosomal peptidyl transferase and inhibitors of the peptidyl transferase A-site function. The 3′-O-AA-derivatives were active acceptors of Ac-Phe in the peptidyl transferase reaction, while the 2′-O-AA-derivatives were completely inactive. Both 2′- and 3′-O-AA-derivatives were potent inhibitors of peptidyl transferase catalyzed Ac-Phe transfer to puromycin. The results indicate that although peptidyl transferase exclusively utilizes 3′-O-esters of tRNA as acceptor substrates, its A-site can also recognize the 3′-terminus of 2′-O-AA-tRNA.

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA.

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.