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

Abstract

Poly(ADP-ribose) polymerase (PARP 4 ) 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.

Manipulating the mammalian genome by homologous recombination

“…PARP knockout mice (147,148) and PARP-deficient cell lines (149) are viable but more sensitive to gamma irradiation. Overexpression of PARP suppresses sister-chromatid exchanges (150), which are caused by HR (151), whereas overexpression of a dominant-negative PARP increases them (152), as does inhibition of PARP by 3-aminobenzamide (153). Inhibition of PARP increases extrachromosomal and intrachromosomal HR up to 5-fold (154)(155)(156) and decreases random integration up to about 100-fold (154,157,158).…”

Poly-ADP-ribosylation in health and disease

“…H-ras transformed NIH3T3 cells and endothelial cells showed loss of tumorigenicity on treatment with PARP inhibitors, benzamide 4-hydroxy-quinazoline or 5-iodo-6-amino-1,2-benzopyrone [52]. Overexpression of a dominantnegative mutant of PARP-1 also reduced the tumorforming ability of HeLa cells with an increase in apoptosis [53]. Although the molecular mechanisms underlying the reduction of tumorigenic potential by PARP-1 inhibition is not fully clarified, a decrease of iNOS expression by PARP-1 inhibition may be involved because a decrease of iNOS expression causes attenuation of cell proliferation and an increase in apoptosis [44].…”

The Genetic Regulation of ADPRT/PARP-1 in Aging and Cancer Susceptibility

“…However, some knockout animals have normal repair of single-strand breaks [Vodenicharov et al, 2000] and normal telomere length [Samper et al, 2001]. Overexpressing the ADPRT/PARP-1 DNA-binding domain did not inhibit DNA replication but led to inhibited poly(ADP-ribosyl)ation [Kupper et al, 1990;Molinete et al, 1993], increased tumor cell apoptosis [Hans et al, 1999], increased polyploidy nuclei [Cayuela et al, 2001], and decreased double-strand break rejoining [Rudat et al, 2001]. However, increased cellular poly(ADP-ribose) accumulation was also observed [Van Gool et al, 1997;Meyer et al, 2000].…”