Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers

Ovarian carcinomas with mutations in the tumour suppressor BRCA2 are particularly sensitive to platinum compounds. However, such carcinomas ultimately develop cisplatin resistance. The mechanism of that resistance is largely unknown. Here we show that acquired resistance to cisplatin can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. First, in a cisplatin-resistant BRCA2-mutated breast-cancer cell line, HCC1428, a secondary genetic change in BRCA2 rescued BRCA2 function. Second, cisplatin selection of a BRCA2-mutated pancreatic cancer cell line, Capan-1 (refs 3, 4), led to five different secondary mutations that restored the wild-type BRCA2 reading frame. All clones with secondary mutations were resistant both to cisplatin and to a poly(ADP-ribose) polymerase (PARP) inhibitor (AG14361). Finally, we evaluated recurrent cancers from patients whose primary BRCA2-mutated ovarian carcinomas were treated with cisplatin. The recurrent tumour that acquired cisplatin resistance had undergone reversion of its BRCA2 mutation. Our results suggest that secondary mutations that restore the wild-type BRCA2 reading frame may be a major clinical mediator of acquired resistance to platinum-based chemotherapy

The chemistry-medicine continuum: Synthetic, computer, spectroscopic and biological studies on new chemotherapeutic leads

This lecture provides an overview of investigations directed towards understanding the molecular mechanism of protein kinase C (PKC) activation and function. Central to this effort are studies on the total synthesis of phorbol, the first asymmetric synthesis of phorbol, and the first synthesis of resiniferatoxin, all involving highly effective applications of [5+2] oxidopyrylium-alkene cycloadditions. The synthesis and affinities of the phorbol ester binding domain of PKC are also presented. In addition, a pharmacophore model for agonist binding to PKC is presented in connection with the design of novel PKC activators. Finally, the computer modeling, NMR structure, synthesis, and biological activity of the first fully synthetic bryostatin analogs are described.close141

Ionizing radiation-induced NF-kappa B activation requires PARP-1 function to confer radioresistance

Recent reports implicate poly(ADP-ribose) polymerase-1 (PARP-1) in the activation of nuclear factor kappaB (NF-κB). We investigated the role of PARP-1 in the NF-κB signalling cascade induced by ionizing radiation (IR). AG14361, a potent PARP-1 inhibitor, was used in two breast cancer cell lines expressing different levels of constitutively activated NF-κB, as well as mouse embryonic fibroblasts (MEFs) proficient or deficient for PARP-1 or NF-κB p65. In the breast cancer cell lines, AG14361 had no effect on IR-induced degradation of IκBα or nuclear translocation of p50 or p65. However, AG14361 inhibited IR-induced NF-κB-dependent transcription of a luciferase reporter gene. Similarly, in PARP-1−/− MEFs, IR-induced nuclear translocation of p50 and p65 was normal, but κB binding and transcriptional activation did not occur. AG14361 sensitized both breast cancer cell lines to IR-induced cell killing, inhibited IR-induced XIAP expression and increased caspase-3 activity. However, AG14361 failed to increase IR-induced caspase activity when p65 was knocked down by siRNA. Consistent with this, AG14361 sensitized p65+/+ but not p65−/− MEFs to IR. We conclude that PARP-1 activity is essential in the upstream regulation of IR-induced NF-κB activation. These data indicate that potentiation of IR-induced cytotoxicity by AG14361 is mediated solely by inhibition of NF-κB activation

Poly(ADP-ribose)polymerases inhibitors prevent early mitochondrial fragmentation and hepatocyte cell death induced by H2O2

Poly(ADP-ribose)polymerases (PARPs) are a family of NAD+ consuming enzymes that play a crucial role in many cellular processes, most clearly in maintaining genome integrity. Here, we present an extensive analysis of the alteration of mitochondrial morphology and the relationship to PARPs activity after oxidative stress using an in vitro model of human hepatic cells. The following outcomes were observed: reactive oxygen species (ROS) induced by oxidative treatment quickly stimulated PARPs activation, promoted changes in mitochondrial morphology associated with early mitochondrial fragmentation and energy dysfunction and finally triggered apoptotic cell death. Pharmacological treatment with specific PARP-1 (the major NAD+ consuming poly(ADP-ribose)polymerases) and PARP-1/PARP-2 inhibitors after the oxidant insult recovered normal mitochondrial morphology and, hence, increased the viability of human hepatic cells. As the PARP-1 and PARP-1/PARP-2 inhibitors achieved similar outcomes, we conclude that most of the PARPs effects were due to PARP-1 activation. NAD+ supplementation had similar effects to those of the PARPs inhibitors. Therefore, PARPs activation and the subsequent NAD+ depletion are crucial events in decreased cell survival (and increased apoptosis) in hepatic cells subjected to oxidative stress. These results suggest that the alterations in mitochondrial morphology and function seem to be related to NAD+ depletion, and show for the first time that PARPs inhibition abrogates mitochondrial fragmentation. In conclusion, the inhibition of PARPs may be a valuable therapeutic approach for treating liver diseases, by reducing the cell death associated with oxidative stress