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F R Althaus

Publications and source records attributed to F R Althaus.

At least 55 records · Page 3Linked to original sources
PubMed1990

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells.

We have determined the ability of UV254nm-irradiated murine lymphoma cells to adapt their NAD+ metabolism to the increased NAD+ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD(+)-utilizing enzyme poly(ADP-ribose) polymerase, NAD+ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV254nm irradiation, a dramatic transient increase of NAD+ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV254nm irradiation, the NAD+ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD+ metabolism to UV254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD+ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells.

Adaptation, Physiological↗
PubMed1990

Differential poly(ADP-ribose) metabolism in repair-proficient and repair-deficient murine lymphoma cells.

The UV254nm-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion into a UV254nm-resistant, non-tumorigenic LY-S subline after prolonged culture in vitro. Here we describe that this conversion leads to distinct changes in several features of intranuclear ADP-ribose polymer metabolism, which may contribute to the altered processing of DNA damage in these cells. The UV254nm-resistant LY-S cells contain 3-fold higher levels of ADP-ribose polymers than LY-R cells. The initial catabolic rate of degradation of these polymers is more than 6-fold higher in LY-S cells. UV254nm irradiation raises the catabolic rates of ADP-ribose polymers in both cell lines. As a consequence, the polymer half-lives decrease from 15 min to 4 min in LY-S cells, and from 96 min to 19 min in LY-R cells. In addition, the rapidly turning over fraction of polymers is much larger in the UV254nm-resistant LY-S cells. These data suggest that the catabolism of poly(ADP-ribose) may be an important factor in the biological expression of DNA damage.

Animals↗
PubMed1990

Uncoupling of DNA excision repair and nucleosomal unfolding in poly(ADP-ribose)-depleted mammalian cells.

The repair of DNA damage in eukaryotic cells is closely coupled with local changes of chromatin structure such that newly synthesized repair patches transiently appear in 'free' DNA domains with increased accessibility to enzymatic and chemical probes. We have isolated these domains from mammalian cells repairing bulky DNA adducts. During the first 3 h of repair, excision of adducts occurred exclusively in free DNA and was closely linked with the appearance of newly synthesized repair patches. Following depletion of chromatin-bound poly(ADP-ribose), the repositioning of repair patches into these domains was completely blocked, although overall repair patch synthesis was unaltered. Concomitantly, DNA adducts were no longer excised and tended to accumulate in free DNA domains. Our results suggest a tight coupling of the excision step with the formation of free DNA domains by a mechanism involving poly ADP-ribosylation of chromatin proteins.

Acetoxyacetylaminofluorene↗
PubMed1990

The poly-ADP-ribosylation system of higher eukaryotes: a protein shuttle mechanism in chromatin?

The role of poly ADP-ribosylation in DNA excision repair was studied in experimental models of various complexities. In intact cells in vivo, the unfolding of chromatin during DNA excision repair apparently requires the presence of a functional poly-ADP-ribosylation system. In vitro studies involving a reconstituted poly-ADP-ribosylation system show that the enzyme poly(ADP-ribose)polymerase has the capacity to shuttle core histones on a core DNA fragment of 146 bp. Under these conditions, the polymerase operates in a strictly processive mode. Furthermore, the polymerase adapts to different shuttling targets by producing very distinct polymer patterns. We conclude that the eukaryotic poly-ADP-ribosylation system has the capacity to regulate DNA-protein interactions and this may be an essential part of the unfolding mechanism of chromatin during excision repair in vivo.

Animals↗
PubMed1989

Poly ADP-ribosylation of proteins. Processivity of a post-translational modification.

The nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) participates in DNA excision repair by post-translational selfmodification ("automodification") and the modification of other chromatin proteins ("heteromodification") with ADP-ribose polymers. We have studied the molecular mechanism of these reactions in a reconstituted in vitro system. After activation by DNA, poly(ADP-ribose) polymerase produces polymers with a distinct size pattern. These polymers are attached to a small subfraction of enzyme molecules. As the reaction progresses, more enzyme molecules are recruited for modification with an identical polymer size pattern. Likewise, the auto- and heteromodification reaction in nucleosomal core particles involves the consecutive addition of a highly conserved polymer size pattern to the acceptor proteins. Thus, a highly conserved polymer size pattern may constitute the molecular signal priming chromatin proteins for a role in DNA excision repair in vivo. The priming reaction is processive.

Animals↗
PubMed1989

Poly(ADP-ribose) catabolism in mammalian cells exposed to DNA-damaging agents.

DNA damage inflicted by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, or by UV254nm, stimulated the catabolism of protein-bound poly(ADP-ribose) in the chromatin of cultured hepatocytes. The stimulation was highest at the largest doses of DNA-damaging treatment. As a consequence, the half-life of ADP-ribosyl polymers may drop to less than 41 s. This rapid turnover contrasts with the slow catabolism of a constitutive fraction of polymers exhibiting a half-life of 7.7 h. Our data suggest that post-incisional stimulation of poly(ADP-ribose) biosynthesis in DNA-excision repair is coupled with an adaptation of poly(ADP-ribose) catabolism in mammalian cells.

Animals↗
PubMed1988

Selective probing of ADP-ribosylation reactions with oxidized 2'-deoxy-nicotinamide adenine dinucleotide.

A homogeneous preparation of an arginine-specific mono(ADP-ribosyl)transferase from turkey erythrocytes effectively utilized 2'-deoxy-NAD+ for the 2'-deoxy(ADP-ribose) modification of arginine methyl ester with an apparent Km of 27.2 microM and a Vmax of 36.4 mumol min-1 (mg of protein)-1. The adduct formed was also used as a substrate by an avian erythrocyte arginine(ADP-ribose)-specific hydrolase that generated free 2'-deoxy(ADP-ribose). In contrast, 2'-deoxy-NAD+ was not a substrate in the initiation or elongation reaction catalyzed by highly purified poly(ADP-ribose) polymerase from calf thymus. However, 2'-deoxy-NAD+ was a potent noncompetitive inhibitor of NAD+ in the elongation reaction catalyzed by the polymerase, with an apparent Ki of 32 microM. These results indicate that 2'-deoxy-NAD+ may be utilized to specifically identify protein acceptors for endogenous mono(ADP-ribosyl)transferases in complex biological systems that may contain a high activity of poly(ADP-ribose) polymerase, i.e., cell nuclei preparations.

ADP Ribose Transferases↗
PubMed1987

Release of core DNA from nucleosomal core particles following (ADP-ribose)n-modification in vitro.

Isolated nucleosomal core particles from rat liver chromatin were ADP-ribosylated in vitro and the consequences of this modification on intranucleosomal DNA-protein interactions were studied by retention gel analysis. A separating force of 11 fN caused the complete release of 146 bp DNA fragments from ADP-ribosylated core particles, while the DNA of unmodified core particles remained protein-associated. We conclude that posttranslational (ADP-ribose)n-modification of chromatin proteins may reduce DNA-protein interactions at the nucleosomal level of chromatin organization.

Adenosine Diphosphate Ribose↗
PubMed1987

Poly(ADP-ribose) may signal changing metabolic conditions to the chromatin of mammalian cells.

In mammalian cells, NAD+ serves a dual role as a respiratory coenzyme and as a substrate for the posttranslational poly(ADP-ribose) modification of chromatin proteins, catalyzed by the nuclear enzyme poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase, EC 2.4.2.30]. Biological evidence strongly suggests that poly(ADP-ribosyl)ation modulates chromatin functions, although the precise molecular mechanisms involved have not yet been elucidated. Here we describe conditions for the rapid uptake of exogenously supplied NAD+ by living hepatocytes in primary monolayer culture. Raising the intracellular NAD+ concentration by 70% caused a 5-fold increase of chromatin-bound poly(ADP-ribose). We conclude that the constitutive level of posttranslational poly(ADP-ribose) modifications of chromatin proteins in mammalian cells is related to the availability of NAD+, which varies in different physiological and pathological states. We propose that poly-(ADP-ribose) may serve a hitherto unrecognized function by signaling altered metabolic conditions to the chromatin and thus modulate its functions in tune with changing metabolic states.

Animals↗
PubMed1986

Poly(ADP-ribose) biosynthesis and suicidal NAD+ depletion following carcinogen exposure of mammalian cells.

Hepatocytes were found to be remarkably resistant to suicidal NAD+ depletion due to consumption for chromatin-associated poly(ADP-ribose) biosynthesis, which normally follows infliction of DNA damage in mammalian cells. N-methyl-N'-nitro-N-nitrosoguanidine treatment, which depleted NAD+ levels of confluent fibroblasts to about 40% of controls, did not reduce hepatocellular NAD+ pools, although poly(ADP-ribose) concentrations were concomitantly elevated by 21-fold. This differential behavior, demonstrable also with other carcinogens, can be attributed to the different NAD+ biosynthetic capacities of these cells.

Animals↗
PubMed1986

A new highly selective physicochemical assay to measure NAD+ in intact cells.

A simple, fast, and highly specific chromatographic method for measuring the content of NAD+ in intact cells has been developed. This procedure involves the separation of NAD+ from the bulk of acid-soluble nucleosides, nucleotides, and other pyridine containing molecules by affinity chromatography on dihydroxyboronyl-Bio-Rex. The boronate purified preparations were utilized for the quantification of NAD+ by strong anion exchange high-pressure liquid chromatography under isocratic conditions using a low salt buffer system. The overall recovery of the method exceeded 80%. This new method was applied to determine the extent of NAD+ consumption in intact hepatocytes following treatment with two different DNA damaging agents. A major advantage of this method is that it allows for the simultaneous determination of poly(ADP-ribose) in the acid-insoluble fraction of the same sample.

Animals↗
PubMed1986

Periodic changes of chromatin organization associated with rearrangement of repair patches accompany DNA excision repair of mammalian cells.

We have used 8-methoxypsoralen to probe the chromatin structure of mammalian cells in situ while they repair pyrimidine dimers or bulky lesions in DNA. We observed that excision repair of these DNA lesions is accompanied by periodic alterations of chromatin organization. In parallel, fluctuations of the rates of repair patch synthesis accompanied these structural changes. Taking advantage of the accessibility of free DNA domains for psoralen intercalation, we have developed a technique to quantitatively isolate the micrococcal nuclease-sensitive, free DNA fraction of native bulk chromatin. We have determined the location of newly synthesized repair patches relative to free DNA domains as a function of repair time. Extensive rearrangements of repair patches from these domains into micrococcal nuclease-resistant DNA were observed. Our results indicate that periodic changes of chromatin organization associated with rearrangement of repair patches accompany the process of excision repair in mammalian cells.

Animals↗
PubMed1986

Tumor-promoting barbiturates act on DNA repair of cultured hepatocytes.

We have observed that two promoters of liver carcinogenesis, i.e. phenobarbital and barbital, markedly increase DNA-repair synthesis of cultured hepatocytes following treatment with the ultimate carcinogens methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, N-acetoxy-2-acetylaminofluorene, and UV light of 254 nm. Phenobarbital also increased the incorporation rates of deoxynucleoside triphosphates into nuclear DNA of permeabilized hepatocytes following carcinogen treatment. The action of these barbiturates apparently correlates with their potential to promote hepatocarcinogenesis in vivo, since the non-promoting agent barbituric acid did not modify carcinogen-induced repair synthesis. Moreover, the mechanisms of action of tumor-promoting barbiturates is different from the known enhancing action on repair synthesis of inhibitors of nuclear poly(ADP-ribose) biosynthesis.

Adenosine Diphosphate Ribose↗
PubMed1983

Consequences of altered poly(ADP-ribose) metabolism on hepatocyte functions in vitro.

Cultured hepatocytes derived from adult rats were used to determine the consequences of altered poly(ADP-ribose)-metabolism on DNA repair and the expression of hepatocellular functions in vitro. Inhibition of poly(ADP-ribose)-biosynthesis affected the process of DNA repair in response to carcinogens at more than one single step. The expression of two fetal enzymes, gamma-glutamyl transpeptidase and the K-(111) type isozyme of pyruvate kinase, was inhibited in hepatocytes in which poly(ADP-ribose)-biosynthesis was suppressed with 3-aminobenzamide or other inhibitors. Our data show that cellular functions as diverse as DNA repair and gene expression are affected by the same experimental manipulations of poly(ADP-ribose)-metabolism in the same cell. This suggests that poly(ADP-ribose)-modification of chromatin proteins affects a mechanism which is involved in the regulation of more than one chromatin function.

Aminobenzoates↗
PubMed1982

ADP-ribosyltransferase activity in cultured hepatocytes. Interactions with DNA repair.

A rapid increase in ADP-ribosyltransferase activity was observed when freshly isolated hepatocytes derived from adult rats were established in primary monolayer culture. (ADP-ribose)n-degrading activity remained constant over a period of 48 h of culture. Inhibition of ADP-ribosyltransferase activity with pyridine derivatives, 3-aminobenzamide, theophylline, or thymidine, was accompanied by an enhanced DNA repair synthesis in response to the direct-acting carcinogen, methyl methanesulfonate, or UV irradiation. Three aminobenzamides differing only in the position of the amino group exhibited the same structure-activity relationship in regard to their action on DNA repair synthesis and ADP-ribosyltransferase. Spermine treatment of hepatocytes apparently had an inverse effect on both these cellular functions. The removal of DNA strand breaks following methyl methanesulfonate treatment was accelerated by inhibitors of ADP-ribosyltransferase. The results suggest that ADP-ribosylation interacts with late stages in the process of DNA repair. This interaction apparently is dependent on the nature of damage imposed on chromatin since repair synthesis in response to a number of carcinogens is unaffected by inhibitors of ADP-ribosyltransferase.

Animals↗