Endogenous Poly ADP-ribose Polymerase Activity Research Articles

Evaluating the Sensitivity of Sporadic Pancreatic Cancer to Poly(ADPribose) Polymerase (PARP) Inhibition (Velaparib, Olaparib, AG14361) as Single Agents and as Chemo-sensitizers

After resection of pancreatic cancer local recurrence occurs in 50%-80% of the cases while metastasis develops 75% of the time. Current, adjuvant therapy often consists of gemcitabine, cisplatin and/or 5-fluorouracil which add a modest increase in median survival by 4-5 months. In this study, we treated human pancreatic cancer cells with poly (ADP-ribose) polymerase (PARP) Inhibitors (AG14361, Veliparib and Olaparib) alone or with gemcitabine, cisplatin or 5-fluorouracil. Methods: CFPAC-1 and BXPC-3, HPAC human pancreatic cancer cell lines were treated for 72 hours with PARP inhibitors alone or in combination with gemcitabine, cisplatin, or 5 – fluorouracil. Validated MTT assays were used to form dose response curves from which the IC50 values were calculated. Results: The PARP1 IC50 values for CFPAC-1, BXPC-3 and HPAC pancreatic cancer cell lines were AG14361 (14.3 µM, 12.7 µM, 38.3 µM), Veliparib (52.6 µM, 100.9 µM 102.0 µM) and Olaparib (79.5 µM, 184.8 µM, 200.2 µM). The IC50 values of cisplatin, were decreased up to 60 fold in the presence of clinically relevant amounts of PARP inhibitor while 5-flourouracil IC50 values were decreased up to 6000 fold in the presence of clinically relevant amounts of PARP inhibitor. Gemcitabine was inhibited up to 73% by PARP inhibitors. Conclusions: Sporadic human pancreatic cancer cells are sensitive to PARP inhibition. PARP inhibitors significantly enhanced the cytotoxicity of cisplatin and 5-fluorouracil while inhibiting gemcitabine. There is little correlation between endogenous PARP activity and the effectiveness of PARP inhibitors.

Poly (ADP-ribose) polymerase1 regulates growth and multicellularity in D. discoideum

Poly (ADP-ribose) polymerase (PARP)-1 regulates various biological processes like DNA repair, cell death etc. However, the role of PARP-1 in growth and differentiation still remains elusive. The present study has been undertaken to understand the role of PARP-1 in growth and development of a unicellular eukaryote, Dictyostelium discoideum. In silico analysis demonstrates ADPRT1A as the ortholog of human PARP-1 in D. discoideum. The present study shows that ADPRT1A overexpression (A OE) led to slow growth of D. discoideum and significant population of AOE cells were in S and G2/M phase. Also, AOE cells exhibited high endogenous PARP activity, significant NAD+ depletion and also significantly lower ADPRT1B and ADPRT2 transcript levels. Moreover, AOE cells are intrinsically stressed and also exhibited susceptibility to oxidative stress. AOE also affected development of D. discoideum predominantly streaming, aggregation and formation of early culminant which are concomitant with reports on PARP's role in D. discoideum development. In addition, under developmental stimuli, increased PARP activity was seen along with developmentally regulated transcript levels of ADPRT1A during D. discoideum multicellularity. Thus the present study suggests that PARP-1 regulates growth as well as the developmental morphogenesis of D. discoideum, thereby opening new avenues to understand the same in higher eukaryotes.

Novel PARP inhibitors sensitize human leukemic cells in an endogenous PARP activity dependent manner

Poly(ADP-ribose) polymerase (PARP) is a critical nuclear enzyme which helps in DNA repair. In this study we report, synthesis and biological studies of novel pyridazine derivatives as PARP inhibitors.

Abstract A102: Synthetic lethality: ABT-888 and carboplatin drug combinations are synergistic for BRCA1- and BRCA2-deficient mouse and human cells in vitro.

Abstract Background: Individuals with an inherited BRCA1 or BRCA2 (BRCA) mutation have an elevated risk of developing breast and ovarian cancer, and this BRCA defect is also present in a subset of sporadic tumors. Tumors do not have homologous recombination repair (HRR) to repair double-stranded breaks (DSBs), but they still can use other DNA repair pathways to survive. Therefore, we hypothesized that crosstalk between DNA repair pathways complements HRR in BRCA-deficient cells, leading to tumor survival. Treatment with either a poly(ADP)ribose polymerase (PARP) inhibitor (PARPi) or a platinum drug induced BRCA-deficient cell lethality by different mechanisms. In addition, combining both drugs has been reported to be synergistic in BRCA mouse models. However, the mechanism of this synergism and whether this synergism occurs in human cells are still unclear. Moreover, not all the patients respond to a PARPi chemotherapy alone. Hence, our objectives were: 1) to study the mechanism by which a PARPi combined with a platinum drug is synthetic lethal in human BRCA1 or BRCA-deficient cells; 2) to assess PARP activity as a biomarker for chemosensitivity. Methods: We compared the cytotoxic effect of ABT-888 (a PARPi) with cisplatin or carboplatin (platinum drugs) alone versus combinations by examining Brca-proficient and -deficient mouse embryonic stem (ES) cell survival using clonogenic assays. The effect of ABT-888 and carboplatin combination treatment on human cell growth was tested in HCC1937 (a triple negative BRCA1-deficient breast cancer line) and EUFA423F (a BRCA2-deficient Fanconi anemia cell line), along with their BRCA complemented lines. Synergism, antagonistic or additive drug interactions were assessed. Drug(s)-induced DSBs, HRR and apoptosis were examined and endogenous PARP activity was determined. Results: Each monotherapy killed or inhibited more Brca/BRCA-deficient cells than the corresponding Brca/BRCA-proficient cells. In Brca-deficient cells, the ABT-888 and carboplatin combination was more synergistic than ABT-888 and cisplatin. All paired BRCA-deficient or complemented cells had the same level of DNA damage following treatment with ABT-888 and/or Carboplatin. BRCA-deficient cells lack HRR, while BRCA-proficient cells use HRR. Failure to repair DSBs in BRCA-deficient cells triggered apoptosis, which was synergistic in the BRCA1-deficient cells. Moreover, higher endogenous PARP activity in Brca/BRCA-deficient cells than their respective Brca/BRCA proficient cells correlated with increased chemosensitivity. Conclusion: Our data suggest that combining ABT-888 and carboplatin will likely be more successful than monotherapy in treating most BRCA-associated cancers. In addition, high endogenous PARP activity could be used as a marker to identify PARPi susceptible tumors. Lack of HRR combined with a strong dependence on PARP activity for repair of platinum lesions led to synthetic lethality in BRCA-deficient cells. A randomized phase II trial has recently been initiated to test this drug combination in patients with BRCA-associated breast cancer and we are currently investigating γH2AX as a pharmacodynamic marker in hair follicles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A102.

The use of PARP inhibitors as single agents and as chemosensitizers in sporadic pancreatic cancer.

e13542 Background: Introduction: After resection of pancreatic cancer local recurrence occurs in 50%-80% of the cases while distant metastasis develops 75% of the time. Most pancreatic cancers have impairments of DNA repair. PARP inhibitors may further inhibit the ability fo the cancer to undergo DNA repair. Current, adjuvant therapy often consist of gemcitabine, cisplatin and/or 5-fluorouracil which add a modest increase in median survival by 4-5 months. In this study, we treated human pancreatic cancer cells with poly(ADP-ribose) polymerase (PARP) Inhibitors (AG14361, veliparib and olaparib) alone or with gemcitabine, cisplatin or 5-fluorouracil. Methods: CFPAC-1 and BXPC-3 , HPAC human pancreatic cancer cell lines were treated for 72 hours with PARP inhibitors alone or in combination with gemcitabine, cisplatin, or 5-fluorouracil. Validated MTT assays were used to form dose response curves from which the IC50 values were calculated. PARP activity was measured indirectly through PAR levels and correlated with the IC50 values of PARP inhibition of each cell line. Results: The PARP1 IC50 values for CFPAC-1, BXPC-3 and HPAC pancreatic cancer cell lines were AG14361 (14.3 µM, 12.7 µM, 38.3 µM ), veliparib (52.6 µM, 100.9 µM 102.0 µM ) and olaparib (79.5 µM, 184.8 µM, 200.2 µM) .These values indicate a sensitivity to PARP1 inhibitors of pancreatic cancer cell lines that is at least as great as the sensitivity of ovarian and breast cancer cell lines to PARP1 inhibition. The IC50 values of cisplatin, were decreased up to 60 fold in the presence of clinically relevant amounts of PARP inhibitor while 5-fluorouracil IC50 values were decreased up to 6000 fold in the presence of clinically relevant amounts of PARP inhibitor. Gemcitabine was inhibited up to 73% by PARP inhibitors. The was poor correlation between PARP activity and teh ability of PARP inhibitors to inhibit pancreatic cancer growth. Conclusions: Sporadic pancreatic cancer cells are exquisitely sensitive to PARP inhibition. PARP inhibitors significantly enhanced the cytotoxicity of cisplatin and 5-fluorouracil while inhibiting gemcitabine. There is little correlation between endogenous PARP activity and the effectiveness of PARP inhibitors to inhibit cancer growth.

Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy

Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP(-/-) fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP(-/-) fibroblasts were exposed for 3 weeks to 20 microM GPI 6150 (1,11b-dihydro-[2H:]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (K(i) = 60 nM) and one of the most potent PARP inhibitors to date (IC(50) = 0.15 microM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by approximately 91%, to about the residual levels in PARP(-/-) cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.

Effects of kainic acid lesioning on poly(ADP-ribose) polymerase (PARP) activity in the rat striatum in vivo.

Poly(ADP-ribose) polymerase (PARP) is activated in glutamate-induced toxicity of neurons in culture (Cosi et al., 1994). Since injection of the excitatory amino acid, kainic acid (KA) into the rat striatum induces a delayed neuronal death, the effects of this in vivo excitotoxin lesioning procedure on striatal PARP activity was investigated. PARP activity was measured in striatal extracts both in the absence ("endogenous" activity) and presence ("total" activity) of exogenously-added fragmented DNA. KA (5nmols/1microl) produced significant and time-dependent changes in striatal PARP activity, compared to saline-injected control animals: no changes at 6h after intrastriatal KA, a 68% and 48% decrease in endogenous and total PARP activity respectively at 12h, a doubling in endogenous PARP activity at 24h, and a 382% and 60% increase in endogenous and total activities at 1 week after KA. PARP cleavage was not detected at any time point. These results suggest a participation of PARP in KA-induced toxicity in the brain in vivo.

Reversal of acquired cisplatin resistance by nicotinamide in vitro and in vivo.

At a concentration of 2.5 mM, nicotinamide (NA), an inhibitor of poly(ADP-ribose) polymerase (PARP), significantly potentiated the cytotoxicity of cisplatin (DDP) in a DDP-resistant rat ovarian tumor cell line (O-342/DDP) in vitro, whereas the same treatment had no substantial effect on DDP's cytotoxic activity against the DDP-sensitive parental line (O-342). Furthermore, in a nude mouse model where the O-342/DDP tumor grew intraperitoneally, whereas DDP given alone at 1 mg/kg x 3 exhibited no antitumor activity as compared with control values due to the resistance, NA given at a nontoxic dose (5 mmol/kg x3) significantly increased the mean survival time (MST) of the tumor-bearing NMRI nude mice from 20.7 days in the DDP-treated group to 29.0 days in the combination group. Mechanism studies showed that endogenous PARP activity (incorporation of tritiated nicotinamide adenine dinucleotide, [3H]-NAD) was 2.6 times higher in O-342/DDP than in O-342 cells and that the presence of 2.5 mM NA during the incubation with the isotope resulted in 73.3% inhibition of the enzyme activity in O-342/DDP cells but in only about 30% inhibition in the sensitive line. However, treatment with NA during and after DDP exposure failed to produce any significant effect on the formation of DNA single-strand breaks (SSB) but decreased the induction of DNA interstrand cross-links (ISCL) by DDP in the sensitive and resistant cell lines. These results suggest that NA might have some clinical potential in reversing DDP resistance, and further studies are therefore warranted to confirm the resistance-reversing effect of NA in other DDP-resistant cell lines.