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The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken together, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 microM PJ-34 for 24 h to obtain the same cytotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment., ((c) 2009 Wiley-Liss, Inc.)

Poly(ADP-ribosyl)ation is a post-translational modification of protein occurring in the nucleus by poly(ADP-ribose) polymerase enzyme activity. The main role of poly(ADP-ribose) polymerase system as "nick sensor" and DNA breaks repair is based on its activation via DNA strand breaks. Furthermore, poly(ADP-ribose) polymerase modifies the binding to DNA of several transcriptional factors by poly(ADP-ribosyl)ation, thereby regulating also transcriptional gene expression. We have analyzed whether poly(ADP-ribose) polymerase activity is involved in basic fibroblast growth factor (FGF2)-mediated upregulation of urokinase-type plasminogen activator (uPA) mRNA. We demonstrated that specific inhibition of poly(ADP-ribose) polymerase activity via 3-aminobenzamide (3ABA) or NAD+ deprivation prevents FGF2-mediated uPA mRNA over-expression and cell-associated plasminogen activator (PA) production in GM7373 endothelial cell line. We verified that FGF2 stimulates poly(ADP-ribose) polymerase activity by a DNA strand breaks-independent manner which involves a mitogen-activated protein kinases (MAPK)-dependent pathway, as confirmed by using PD98059 inhibitor and anisomycin stimulation. Poly(ADP-ribose) polymerase involved in this mechanism is mainly the 60 kDa molecular mass isoform, that presents an increase in serine phosphorylation in the presence of FGF2., (2005 Wiley-Liss, Inc.)

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G1-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G1-S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration-linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.