Jan-Heiner Küpper

Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

The zinc‐finger DNA‐binding domain (DBD) of poly (ADP‐ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA‐damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP‐ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc‐finger, were overproduced in Escherichia coli, in CV‐1 monkey cells or in human fibroblasts to study their DNA‐binding properties, the trans‐dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA‐binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). Furthermore, overproduced wild‐type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc‐finger of PARP for DNA single‐stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.

Glucocorticoid Hormone Stimulates Mitochondrial Biogenesis Specifically in Skeletal Muscle

High levels of circulating glucocorticoid hormone may be important mediators for elevating resting metabolic rate upon severe injury or stress. We therefore investigated the effect of dexamethasone on mitochondrial biogenesis in rats (6 mg/kg daily) as well as in cells in culture (1 microM) over a period of 3 d. A marked stimulation of mitochondrial DNA transcription and increased levels of cytochrome c oxidase activity were found in skeletal muscle of rats and differentiated mouse C2C12 muscle cells, but not in other tissues, myoblasts, or other cell lines. The effect was inhibited by RU486. Therefore, increased occupancy of glucocorticoid receptors is necessary, but not sufficient to increase mitochondrial biogenesis and other, skeletal muscle specific factors are postulated. Expression of the mitochondrial transcription factor A was unchanged, suggesting a possible involvement of the recently described mitochondrial glucocorticoid receptor. Expression of uncoupling protein-3 was also unchanged. In conclusion, our results show that high levels of glucocorticoid hormone are sufficient to stimulate mitochondrial biogenesis; however, only in skeletal muscle. Increased mitochondrial mass in this tissue, without changes of the coupling state of the respiratory chain, might be the molecular basis for the elevated resting metabolic rate observed under high cortisol levels in humans.

DNA Excision Repair and DNA Damage-Induced Apoptosis Are Linked to Poly(ADP-Ribosyl)ation but Have Different Requirements for p53

ABSTRACT Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD+ to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.

Negative regulation of alkylation‐induced sister‐chromatid exchange by poly(ADP‐ribose) polymerase‐1 activity

One of the earliest responses to DNA damage in eukaryotic cells is activation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), a DNA strand break–dependent nuclear enzyme which covalently modifies proteins with poly(ADP‐ribose). Here, we show that conditional over‐expression of PARP‐1 in stably transfected hamster cells, which causes cellular over‐accumulation of poly(ADP‐ribose) by several‐fold, strongly suppresses alkylation‐induced sister‐chromatid exchange (SCE), while cytotoxicity of alkylation treatment is slightly enhanced. Viewed together with the known potentiation of SCE by abrogation of PARP‐1 activity, our results provide evidence that PARP‐1 activity is an important regulator of alkylation‐induced SCE formation, imposing a control that is strictly negative and commensurate with the level of enzyme activity. Int. J. Cancer 88:351–355, 2000.

Direct stimulation of poly(ADP ribose) polymerase in permeabilized cells by double-stranded DNA oligomers.

Poly(ADP ribosyl)ation, a post-translational modification of nuclear proteins catalyzed by poly (ADP ribose) polymerase, is an immediate response of most eukaryotic cells to DNA strand breaks and has been implicated in DNA repair and other cellular phenomena associated with DNA strand breakage. Poly(ADP ribose) polymerase activity levels have been frequently assayed by incubating permeabilized cells with radioactively labeled NAD+ as substrate. In such assays enzyme activation has routinely been achieved indirectly by prior exposure of living cells to carcinogens or by adding DNase I to permeabilized cells, thereby introducing strand breaks in chromosomal DNA. Here we show that, as an alternative method, the direct activation of purified poly(ADP ribose) polymerase by double-stranded oligonucleotides (N. A. Berger and S. I. Petzold, 1985, Biochemistry 24, 4352-4355) can be adopted for permeabilized cell systems. The inclusion of a palindromic decameric deoxynucleotide in the reaction buffer stimulated the enzyme activity in permeabilized Molt-3 human lymphoma cells up to 30-fold (at 50 micrograms/ml [corrected] oligonucleotide concentration) in a concentration-dependent manner. The activating effect of oligonucleotides was also evident when ethanol-fixed HeLa cells were postincubated with NAD+ to allow poly(ADP ribose) synthesis to occur in situ, which was detected as specific anti-poly (ADP ribose) immunofluorescence. We conclude that double-stranded oligonucleotides can be conveniently used as chemically and stoichiometrically well-defined poly (ADP ribose) polymerase activators in permeabilized or ethanol-fixed mammalian cells.

DETECTION OF POLY(ADP-RIBOSE) POLYMERASE AND ITS REACTION PRODUCT POLY(ADP-RIBOSE) BY IMMUNOCYTOCHEMISTRY

SummaryPoly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD+ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.

Comparative analysis of virus–host cell interactions of haemagglutinating and non-haemagglutinating strains of coxsackievirus B3

Decay-accelerating factor (DAF/CD55), and coxsackievirus-adenovirus receptor (CAR) have been identified as cellular receptors for coxsackie B viruses (CBV). To elucidate the interplay of DAF and CAR on the cell surface, virus-receptor interactions of two coxsackieviruses of serotype B3 (non-haemagglutinating CBV3 and haemagglutinating CBV3-HA strain) were analysed. Binding assays revealed clear differences between these viruses with regard to their interactions with DAF and CAR. However, only the combination of anti-DAF and anti-CAR antibodies resulted in complete inhibition of virus binding for both strains. In plaque-reduction assays, anti-DAF antibodies had no effect, whereas CAR-specific antibodies significantly reduced productive infection of HeLa cells by both viruses. Interestingly, a synergistic inhibitory effect of anti-DAF and anti-CAR antibodies was also observed with regard to infection. These findings support the model of preferential interactions of both strains of CBV3 with closely associated DAF and CAR proteins on HeLa cells, despite displaying clear differences in their binding phenotypes.

Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: evidence for increased tumor cell apoptosis in vivo.

Poly(ADP-ribose) polymerase (PARP4) catalyzes the formation of ADP-ribose polymers covalently attached to proteins by using NAD+ as substrate. PARP is strongly activated by DNA single- or double-strand breaks and is thought to be involved in cellular responses to DNA damage. We characterized a dominant negative PARP mutant, i.e. the DNA-binding domain of this enzyme, whose overexpression in cells leads to increased genetic instability following DNA damage. In order to study whether PARP activity is also implicated in the process of tumorigenesis, we generated stably transfected HeLa cell clones with constitutive overexpression of dominant negative PARP and investigated tumor formation of these clones in nude mice. We found that inhibition of PARP activity dramatically reduces tumor forming ability of HeLa cells. Moreover, we provide strong evidence that the observed reduction in tumor forming ability is due to increased tumor cell apoptosis in vivo. Viewed together, our data and those from other groups show that inhibition of PARP enzyme activity interferes with DNA base excision repair and leads to increased genetic instability and recombination but, on the other hand, can sensitize cells to apoptotic stimuli and by this mechanism may prevent tumor formation.

Growth inhibition of cervical cancer cells by the human retinoic acid receptor β gene

Transcription of the retinoic receptor β (RARβ) gene is activated in a ligand‐dependent manner by the retinoic acid receptor α. Reduced RARβ gene expression and loss of ligand inducibility are frequently observed in human carcinoma cells indicating that such alterations might contribute to carcinogenesis. In this study we have analyzed the influence of RARβ on cervical cancer cell growth. Transfection of HeLa cells with RARβ expression plasmids resulted in reduced clonal cell growth in the presence of retinoic acid (RA). RA‐induced growth inhibition in HeLa × fibroblast hybrid cells was partially relieved by a dominant‐negative RARβ mutant. HeLa clones stably expressing a RARβ transgene under control of the human β‐actin promoter [HeLa(RARβ)] were established and analyzed for transgene‐mediated growth alterations in vitro and in vivo. Anchorage‐independent growth of the HeLa(RARβ) lines was indistinguishable from that of control cells in the absence of RA, but strongly impaired after RA treatment. Reduced tumor growth of HeLa(RARβ) clones was associated with high RARβ protein levels. Somatic cell fusion experiments revealed that the loss of ligand inducibility of RARβ gene expression in HeLa cells cannot be complemented by fusion with other cervical cancer cell lines. Our data indicate, firstly, that RARβ is a negative regulator of tumor cell growth and, secondly, that cancer‐associated defects in RARβ gene expression are caused by stable, non‐complementable silencing mechanisms. Int. J. Cancer 85:289–295, 2000. ©2000 Wiley‐Liss, Inc.

Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates alkylation-induced shuttle-vector mutagenesis in Chinese hamster cells

In most eukaryotic cells, the catalytic activation of poly(ADP-ribose) polymerase (PARP) represents one of the earliest cellular responses to the infliction of DNA damage. To study the biological function(s) of poly(ADP-ribosyl)ation, we have established stable transfectants (COM3 cells) of the SV40-transformed Chinese hamster cell line C060 which conditionally overexpress the PARP DNA-binding domain upon addition of dexamethasone. We could demonstrate that DNA-binding domain overexpression, which leads to trans-dominant inhibition of poly(ADP-ribosyl)ation, potentiates the cytotoxicity of alkylation treatment and of γ-radiation [21]. Likewise, carcinogen-induced gene amplification, viewed as a manifestation of genomic instability, was potentiated by the overexpression of the PARP DNA-binding domain [22]. Recently, we studied the effect of trans-dominant PARP inhibition on mutagenesis by employing a shuttle-vector assay in which mutagen-exposed plasmid pYZ289 is electroporated into COM3 cells. We could show that dexamethasone-induced overexpression of the PARP DNA-binding domain in COM3 cells potentiates the mutagenicity of the alkylating agent N-methyl-N-nitrosourea, while no effect of dexamethasone treatment on mutation frequency was recorded in control cells lacking the PARP DNA-binding domain transgene. Taken together, our results further substantiate the role of poly(ADP-ribosyl)ation in the maintenance of genomic integrity and stability under conditions of genotoxic stress.