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.

Clustering of pericentromeres initiates in step 9 of spermiogenesis of the rat (Rattus norvegicus) and contributes to a well defined genome architecture in the sperm nucleus.

Fluorescence in situ hybridization with centromeric, telomeric and whole chromosome paint probes was used to study nuclear topology in epididymal sperm as well as spermatids from testis tissue sections of the rat. Pericentromeric regions of 9 chromosomes of the rat (n=21) were labeled with a satellite I specific DNA probe. Pericentromeres showed few tandem associations in spermatids of steps 1-8 of spermiogenesis. At step 9, pericentromeric regions associated to form an elongated cluster in the spermatid nucleus. This arrangement was also seen in the sperm nucleus. FISH with telomere probes revealed numerous, variably arranged signals in round and elongated spermatids as well as sperm nuclei. Telomere signals showed a tendency for pairwise association which was more pronounced in elongated spermatid and epididymal sperm nuclei. FISH to DTT treated sperm suggested that telomeres reside at the periphery and that pericentromeres are located in the nuclear interior. Chromosome painting with rat chromosome 2 and 12 specific microdissection library probes showed that these chromosomes predominantly occupy compact and variably shaped territories during spermatid maturation. In elongated epididymal sperm nuclei chromosome 2 and 12 territories took up specific positions. We suppose that the associations of pericentromeres during step 9 render a well defined nuclear topology which facilitates the ordered compaction of the genome at subsequent stages.