Mitochondria play a central role in programmed cell death through both apoptotic and necrotic signals (Gross et al

Polycyclic aromatic hydrocarbons (PAH) are one of the major carcinogens in tobacco smoke. They are metabolically activated through different routes to form either diol-epoxides, PAH o-quinones, or radical cations and each of which has been proposed to be an ultimate carcinogen. To study how PAH metabolites mutate p53, we used a yeast reporter gene assay based on the p53 transcriptional activity. Colonies expressing wt p53 turn white (ADE +) and those expressing mutant p53 turn red (ADE −). We examined the mutagenicity of three o-quinones, benzo[a]pyrene-7,8-dione, benz [a] anthracene-3,4-dione and dimethylbenz [a]anthracene-3,4-dione, and compared them with (±) antibenzo[a]pyrene diol epoxide ((±)-anti-BPDE) within the same system. The PAH o-quinones tested gave a dose-dependent increase in mutation frequency in the range of 0.160 -0.375 μM quinone, provided redox-cycling conditions were used. The dominant mutations were G to T transversions (>42%), and the incidence of hotspot mutations in the DNA-binding domain was more than twice than would be expected by a random distribution. The dependence of G to T transversions on redoxcycling implicates 8-oxo-dGuo as the lesion responsible, which is produced under identical conditions (Chem. Res. Toxicol. (2005) 18: 1027). A dose-dependent mutation frequency was also observed with (±)-anti-BPDE but at micromolar concentrations (0-20 μM). The mutation pattern observed was G to C (63%) > G to A (18%) > G to T (15%) in umethylated p53 and was G to A (39%) > G to C (34%) > G to T (16%) in methylated p53. The preponderance of G mutations is consistent with the formation of anti-BPDE-N 2 -dGuo as the major adduct. The frequency of hotspots mutated by (±)-anti-BPDE was essentially random in umethylated and methylated p53, suggesting that 5′-CpG-3′ islands did not direct mutations in the assay. These data suggest that smoking may cause mutations in p53 by formation of PAH o-quinones which produce reactive oxygen species. The resultant 8-oxo-dGuo yields a pattern of mutations but not a spectrum consistent with that seen in lung cancer; we suggest that the emergence of the spectrum requires biological selection. KeywordsAldo-keto reductase; polycyclic aromatic hydrocarbon; ortho-quinones; reactive oxygen species Supporting Information Available: Tables of supplemental data S1-S6. These tables list the mutations observed for each PAH o-quinone, BPDE and MNNG in unmethylated or methylated p53 cDNA by codon number, the nucleotide change observed, the sequence context (5′ and 3′-codons), the type of mutation, and the amino acid substitution. This material is available free of charge via the internet at

PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metabolically converted into DNA-reactive metabolites. P4501A1/P4501B1 plus epoxide hydrolase activate PAH to (+/-)- anti-benzo[ a]pyrene diol epoxide ((+/-)- anti-BPDE), which causes bulky DNA adducts. Alternatively, aldo-keto reductases (AKRs) convert intermediate PAH trans-dihydrodiols to o-quinones, which cause DNA damage by generating reactive oxygen species (ROS). In lung cancer, the types or pattern of mutations in p53 are predominantly G to T transversions. The locations of these mutations form a distinct spectrum characterized by single point mutations in a number of hotspots located in the DNA binding domain. One route to the G to T transversions is via oxidative DNA damage. An RP-HPLC-ECD assay was used to detect the formation of 8-oxo-dGuo in p53 cDNA exposed to representative quinones, BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione under redox cycling conditions. Concurrently, a yeast reporter system was used to detect mutations in the same cDNA samples. Nanomolar concentrations of PAH o-quinones generated 8-oxo-dGuo (detected by HPLC-ECD) in a concentration dependent manner that correlated in a linear fashion with mutagenic frequency. By contrast, micromolar concentrations of (+/-)- anti-BPDE generated (+)- trans- anti-BPDE-N (2)-dGuo adducts (detected by stable-isotope dilution LC/MS methodology) in p53 cDNA that correlated in a linear fashion with mutagenic frequency, but no 8-oxo-dGuo was detected. Previous studies found that mutations observed with PAH o-quinones were predominately G to T transversions and those observed with (+/-)- anti-BPDE were predominately G to C transversions. However, mutations at guanine bases observed with either PAH-treatment occurred randomly throughout the DNA-binding domain of p53. Here, we find that when the mutants were screened for dominance, the dominant mutations clustered at or near hotspots primarily at the protein-DNA interface, whereas the recessive mutations are scattered throughout the DNA binding domain without resembling the spectra observed in cancer. These observations, if extended to mammalian cells, suggest that mutagenesis can drive the pattern of mutations but that biological selection for dominant mutations drives the spectrum of mutations observed in p53 in lung cancer.

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilatorstimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.Endothelial cell motility and morphology are tightly regulated processes (1, 2). However, in some diseases including cancer, age-related macular degeneration, diabetic retinopathy, and rheumatoid arthritis, control of these processes is lost resulting in improper endothelial vascularization (angiogenesis) (3). Consequently understanding the molecular mechanisms and signals that regulate endothelial cell motility and morphology may lead to novel therapeutic strategies for these conditions. The Rho family of small GTPases regulates cell proliferation, apoptosis, motility, and morphology (4 -6). Rac and Cdc42 are two of the best characterized Rho GTPase family members, and one of their shared effectors is the serine/threonine p21-activated kinase (Pak) 2 (7). Signals as varied as insulin, estrogen, glial growth factor, interleukin-3, or angiogenic factors activate and regulate Pak (8 -13). In endothelial cells, Pak is stimulated by vascular endothelial growth factor, basic fibroblast growth factor, and angiopoietins 1 and 2 to regulate angiogenesis, motility, and cell survival (8,13,14). Microinjection of mutant forms of Pak such as dominant negative mutants or an inhibitory peptide called the Pak PID (Pak inhibitory domain, amino acids 83-149) reduces motility ...