Klaus Wielckens

Reduction of DNA-polymerase β Activity of CHO Cells by Single and Combined Heat Treatments

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.

Subcellular distribution of mono(ADP-ribose) protein conjugates in rat liver

Summary Rat liver nuclei isolated by neutral sucrose or citric acid procedures contain 2 OH-resistant mono(ADP-ribosyl) protein conjugates were associated with the mitochondrial fraction, and, to a small degree, with the plasma membranes. The NH 2 OH-sensitive conjugates were primarily found in the fractions containing the endoplasmic reticulum. This distribution is in accordance with multiple and independent functions of mono ADP-ribosylation and poly ADP-ribosylation reactions.

[49] Quantification of protein-bound ADP-ribosyl and (ADP-ribosyl)n residues

Publisher Summary Proteins modified by single adenosine 5′-diphosphoribose (ADPR) groups [mono(ADPR)conjugates] and by polymeric ADPR residues [poly(ADPR)conjugates] are found in many eukaryotic cells. These ADPR protein conjugates can be further divided in those that are sensitive to neutral 0.4 M hydroxylamine (NH 2 OH-sensitive conjugates) and others that are hydroxylamine resistant. This chapter describes procedures for the determination of mono(ADPR) and poly(ADPR) residues in protein conjugates which are based on radioimmunoassays. Protein-bound mono(ADPR) groups are released from the TCA-insoluble tissue fraction either by neutral NH 2 OH (hydroxylamine-sensitive ADPR protein conjugates) and subsequently converted to 5′-AMP by treatment with alkali, or by NaOH, which converts hydroxylamine-sensitive plus hydroxylamine-resistant ADPR protein conjugates directly to 5′-AMP. Radioimmunoassay for determination of protein-bound mono(ADPR) residues is based on highly specific anti-5′-AMP antibodies. It allows the determination of ADPR residues in TCA insoluble tissue fractions. On the other hand, protein-bound oligo and poly(ADPR) chains are released by treatment with alkali, which leaves the polymer intact but converts mono(ADPR) to 5′-AMP.

Mono ADP-ribosylation and poly ADP-ribosylation of proteins in normal and malignant tissues.

Three subclasses of (ADPR)n protein conjugates were quantified from intact tissue; proteins carrying poly(ADPR) and two types of mono(ADPR) protein conjugates, one susceptible, the other resistant to neutral hydroxylamine. Mono(ADPR) conjugates were found in all major compartments of the liver cell although the two subfractions were unevenly distributed. Poly(ADPR) protein conjugates appear to be restricted to the nucleus. Independent changes of the subclasses in normal and malignant tissues associated with cell growth and differentiation also point to independent functions. Hydroxylamine-resistant mono(ADPR) protein conjugates of various tissues changed with the degree of terminal differentiation. Formation of poly(ADPR) proteins, on the other hand, was stimulated by treatment of cells with alkylating agents which lead to DNA-fragmentation. This points to an involvement of polyADP-ribosylation in DNA excision repair.

Quantification without purification of blood and tissue adenosine by radioimmunoassay.

Highly specific anti-adenosine antibodies were produced in rabbits by the injection of N6-carboxymethyl adenosine-methylated serum albumin conjugates. They were used to develop a radioimmunoassay allowing the quantitation of adenosine in the range 0.1-10 pmol per sample. Inosine did not interfere except at 300 times higher concentrations, while AMP (ATP) did not displace the [3H]adenosine tracer even at 10(5) (10(6) ) times higher amounts. Due to the high specificity of the anti-adenosine antibodies, determination of blood and tissue adenosine levels could be performed directly from perchloric acid extracts. Values for human peripheral venous blood from various donors obtained with this procedure varied between 46 and 148 pmol/ml blood. The procedure was also applied to HeLa cultures with low and high intracellular adenosine. The reliability of the method was demonstrated by comparative analyses using HPLC purification of adenosine prior to the radioimmunoassay.

Glucocorticoid-induced lymphoma cell death: The good and the evil

Glucocorticoid hormones and their synthetic derivatives are widely used in therapy due to their strong anti-inflammatory and immunosuppressive potential. While the molecular basis of the anti-inflammatory action is to date at least partially understood, knowledge regarding the mechanism underlying glucocorticoid effects on the immune system is rather fragmentary. The immunosuppression could be attributed to at least two distinct processes: inhibition of the production of growth mediators and glucocorticoid-induced cell death. The mechanism of glucocorticoid-induced cell death can be divided into two steps, a reversible growth inhibition and cell lysis. The first step is characterized by many metabolic alterations typical of the catabolic potential of corticosteroids. After a delay of several hours activation of an endonuclease appears to initiate the lytic phase. By the action of this endonuclease the DNA is fragmented. In opposition to the chromatin damage, poly(ADP-ribosyl)ation is activated in order to stabilize the chromatin structure until the antagonistic potential is exhausted and the cells die. Therefore it can be speculated that the lethal event in glucocorticoid-induced cell death is a destruction of the regular chromatin structure.

ADP-ribosylation of nuclear proteins.

Covalent modification of nuclear proteins by mono ADP-ribosylation and poly ADP-ribosylation was studied in various tissues and under various growth conditions with the aid of a newly developed radioimmunoassay. Two types of (ADPR)n protein conjugates were found in vitro and in intact tissues which could be differentiated by their sensitivity towards neutral NH2OH. Analysis of the cell cycle of Physarum polycephalum showed independent synthesis of these two fractions. Widely differing ratios of the two types of conjugates were also found in different hepatic tissues. Yoshida hepatoma cells had very low levels of both types of protein bound ADPR residues (1 ADPR residue per 2,000–3,000 DNA bases), while neonatal liver was characterized by the (near) absence of NH2OH resistant ADPR protein conjugates. Conjugates carrying poly(ADPR) residues exhibited independent variations. Proliferating tissues consistently had somewhat lower levels of mono(ADPR) protein conjugates than the same tissues in resting conditions. The independent changes of mono and poly(ADPR) protein conjugates under various conditions, and the existence of multiple acceptor proteins in the nucleus point to multiple functions of ADP ribosylation rather than to a single role of this covalent modification reaction. Analysis of (ADPR)n histone H1 conjugates isolated from (3H) adenosine labeled HeLa cultures by a new procedure indicated modification of < 1% of total histone H1 by ADP-ribosylation. Most of the conjugates carried single ADPR residues, while (ADPR)n histone H1 conjugates formed by incubation of isolated nuclei with NAD contained mainly poly(ADPR) residues. There were additional basic differences in the degree of ADP- and polyADP-ribosylation as well as in the types of bonds linking ADPR to the histone which seriously limit the conclusions drawn from experiments with isolated nuclei or chromatin. The low levels of ADPR conjugates in vivo, together with the high enzymic capacity for ADPR transfer observed in many cell types, point to a rapid turnover of protein bound ADPR residues being consistent with the occurrence of rapid transient modifications of protein in localized regions of chromatin.

Mechanisms of Glucocorticoid-Induced Growth Inhibition and Cell Lysis in Mouse Lymphoma Cells

Glucocorticoid hormones are known to have a wide variety of molecular effects by induction or repression of proteins at the transcriptional level via a receptor system. Glucocorticoids, depending on the nature of the target tissue, can not only regulate carbonhydrate, protein and nucleic acid metabolism but can also accelerate or inhibit cellular growth or differentiation. Among the cell types that involute during prolonged exposure to steroids are certain lymphocytes and lymphoma cells. This effect is the basis of steroid therapy for malignant lymphomas. The steroid-induced death of lymphoma cells has morphological characteristics clearly distinct from necrosis, i.e., early alterations of the nuclear structure together with progressive reduction of the cellular volume but preservation of the integrity of cytosolic organelles [1] which are altered early during necrosis. This second type of cell death, called apoptosis, represents an active process involving an alteration of the pattern of active genes [2] in contrast to necrosis which is the consequence of environmental perturbations.

ADP-Ribosylation of Proteins in Normal and Neoplastic Tissues

In 1966, Mandel and coworkers in Strasbourg described an enzyme located in the nucleus of chicken liver, which is able to transfer active ADPR groups from NAD to form a homopolymer (1). In this polymer ADPR residues are linked O-glycosidically. Shortly later, Hayaishi and coworkers (2) as well as Sugimura’s group (3) reported on similar findings. Recent evidence indicates that the structure can also be branched (4). The nuclear ADPR transferase is apparently present in all eukaryotes (5–7). It differs basically from ADPR transferase subsequently found to be associated with certain bacterial toxins and with phages: The prokaryotic enzymes transfer single ADPR residues either to one acceptor protein as in the case of Diphteria toxin, where the ribosomal elongation factor 2 is the target (8), or to multiple proteins which serve as acceptors of the T4 and the N4 phage-induced ADPR transferase (cf. 5–7). By contrast, the nuclear enzyme forms preferentially oligo- and poly(ADPR) conjugates with various protein acceptors.

Mono ADP-ribosylation and Poly ADP-ribosylation of Proteins

Postsynthetic modification of proteins by transfer of ADP-ribosyl groups from NAD has been shown to occur in numerous systems. Besides ADPR transferase reactions associated with the action of bacterial toxins and viruses, ADP ribosylation reactions were also observed in eukaryotic cells (cf. Hilz and Stone 1976; Hayaishi and Ueda 1977; Purnell et al. 1980).