Efficacy of osimertinib for the treatment of previously EGFR TKI treated NSCLC patients: a meta-analysis.

BACKGROUND: This study evaluates the efficacy of osimertinib for the treatment of previously epidermal growth factor receptor tyrosine kinase inhibitors (EFGR-TKI) treated non-small cell lung cancer (NSCLC) patients. METHOD: Research articles reporting the efficacy of osimertinib for NSCLC patients were identified from literature databases (Embase, Ovid, PubMed and Scopus) by following pre-determined eligibility criteria. Response and survival data were extracted from study reports and were pooled under random-effects model to obtain overall/subgroup effect sizes of selected efficacy outcomes. RESULTS: Nine studies (950 patients; age 60.1 years [95% confidence interval: 57.2, 63.1]; 65% [95% CI: 62, 69] females; 69% [35, 100] with T790M; 61% [53, 68] with ex19del; and 35% [29, 41] with L858R mutations). Osimertinib treatment was associated with a PFS of 11.17 months [7.80, 14.55] which was longer in treatment-naïve (20.30 [15.37, 25.23]) than in prior EGFR-TKI-treated (10.20 [9.60, 10.80]) patients. 1-year survival was 81.29% [73.25, 89.32]. Complete response rate was 1.48% [1.19, 1.76]. PR was achieved in 53.18% [24.18, 82.18] patients which differed between treatment-naïve and prior EGFR-TKI-treated patients (74.48 [65.59, 83.37] and 67.99% [62.68, 73.30], respectively. Objective response rate and disease control rates were 69.80% [64.84, 74.77] and 92.43% [89.42, 95.43], respectively, which did not differ between treatment-naïve and prior EGFR-TKI-treated patients. CONCLUSION: Osimertinib treatment yields approximately 10 months PFS in prior EGFR-TKI-treated and 20 months in treatment-naïve NSCLC patients. Partial response rate is also higher in treatment-naïve patients. However, objective response rate (ORR) and disease control rate (DCR) did not differ between groups of patients.

[Prediction model for survival in patients with biliary tract cancer: a development and validation study].

Objective: The aim of the present study was to investigate the survival rate and its prognostic factors for patients with biliary tract cancer, and then a prognostic risk prediction model was constructed to predict the survival probability of patients. Methods: A total of 14 005 patients with biliary tract cancer (including gallbladder cancer, extrahepatic bile duct cancer, and ampulla of Vater cancer), who were diagnosed between 2010 and 2015 in the US National Cancer Institute Surveillance, Epidemiology, and End Results Program (SEER) were included in the development cohort. The prognostic risk factors of biliary tract cancer were investigated using multivariate Cox regression models. The predictive nomograms were then constructed to predict the overall survival probability of 1, 3, and 5 years, and the predictive discrimination and calibration ability of the nomograms were further evaluated. Meanwhile, 11 953 patients who were diagnosed during 2004 to 2009 from SEER Program were then selected to validate the external predictive accuracy of the prediction models. Results: The 1, 3 and 5-year cumulative survival rates of patients with biliary tract cancer were 41.9%, 20.4% and 15.3%, respectively, in the development cohort. Age greater than 50 years, African Americans and Native Americans and Alaska Natives, higher T, N and M stage and poor histological differentiation grade were risk factors for death, while married status, Asia-Pacific Islanders, insured status and surgery on primary site were protective factors. Gender was not significantly associated with the overall survival. The C statistic of the prediction model was 0.73 (95%CI: 0.72-0.74), and the calibration curve showed that the interaction curves of predictive and actual survival rates of 1, 3 and 5 years were close to the 45 degree diagonal. Results in the validation cohort were similar with those in the construction cohort, with a C statistic of 0.70 (95%CI: 0.69-0.72), indicating high external applicability of the prediction model. Findings from gallbladder cancer, extrahepatic bile duct cancer, and ampulla of Vater cancer are in consistent with the overall biliary tract cancer. Conclusions: The survival rate of patients with biliary tract cancer is relatively poor, and the survival prediction model based on prognostic factors has high prediction accuracy. In the future, this prognostic prediction model could be applied to clinical practice to guide individualized treatment for patients with biliary tract cancer.

Prevalence of diastolic dysfunction in patients with ankylosing spondylitis: a cross-sectional study.

OBJECTIVES: To determine the prevalence of diastolic dysfunction (DD) in patients with ankylosing spondylitis (AS) by following recommended criteria from the American Society of Echocardiography (ASE) and using single variables reflecting DD. METHOD: A total of 187 patients with AS (105 men; mean age 51 ± 13 years; mean duration of disease 15 ± 11 years) fulfilled the inclusion criteria and underwent pulsed-wave and tissue Doppler imaging. RESULTS: By following ASE recommended criteria, we observed that 12% of patients with AS had mild DD. We also compared single standard Doppler values with normal age-stratified reference values and showed a wide variation in the number of patients with AS outside the 95% confidence interval (CI) of normal values depending on the variable chosen (ranging from 1.1% to 30.5%). CONCLUSIONS: By following recommended criteria, our cross-sectional study shows that DD was infrequent and mild in patients with AS.

The concept of two-dimensional electrophoresis-guided purification proven by isolation of rhodocetin from Calloselasma rhodostoma (Malayan pit viper)

Two-dimensional gel electrophoresis (2DE) is an important tool for investigating the complexity of snake venom proteomes. Apart from applications based on whole proteome analysis, we suggest that 2DE can be used as an assay to guide the progress of protein purification. The aim of this study was to prove the feasibility of this concept by using it to purify rhodocetin from Calloselasma rhodostoma venom. Rhodocetin (α subunit) spot on the 2DE profile of C. rhodostoma venom was first identified and confirmed by mass spectrometry, with a molecular mass of 16 kDa and calculated pI of 5.16. Rhodocetin was subsequently purified by successive anion-exchange and gel filtration chromatography. Every peak from both chromatography profiles was collected and tested on 2DE. The presence of rhodocetin (α subunit) spot in the 2DE profile of the peak DP2 indicated the presence of the protein. The purified compound was used to spike the crude venom. A spiked spot with a 1.6-fold increase in intensity was observed and its position matched to that of rhodocetin (α subunit) on the 2DE profile. Together, these spots confirmed the identity of the purified compound as rhodocetin. Hence, our results have demonstrated the effectiveness of the concept we now term 2DE-guided purification.(AU)

Downregulation of miR-205 and miR-31 confers resistance to chemotherapy-induced apoptosis in prostate cancer cells.

Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2'-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer.

A crossed beams study of the reaction of carbon atoms, C(3Pj), with vinyl cyanide, C2H3CN(X 1A')--investigating the formation of cyano propargyl radicals.

The chemical dynamics of the reaction of ground state carbon atoms, C(3Pj), with vinyl cyanide, C2H3CN(X 1A'), were examined under single collision conditions at collision energies of 29.9 and 43.9 kJ mol(-1) using the crossed molecular beams approach. The experimental studies were combined with electronic structure calculations on the triplet C4H3N potential energy surface (H. F. Su, R. I. Kaiser, A. H. H. Chang, J. Chem. Phys., 2005, 122, 074320). Our investigations suggest that the reaction follows indirect scattering dynamics via addition of the carbon atom to the carbon-carbon double bond of the vinyl cyanide molecule yielding a cyano cyclopropylidene collision complex. The latter undergoes ring opening to form cis/trans triplet cyano allene which fragments predominantly to the 1-cyano propargyl radical via tight exit transition states; the 3-cyano propargyl isomer was inferred to be formed at least a factor of two less; also, no molecular hydrogen elimination channel was observed experimentally. These results are in agreement with the computational studies predicting solely the existence of a carbon versus hydrogen atom exchange pathway and the dominance of the 1-cyano propargyl radical product. The discovery of the cyano propargyl radical in the reaction of atomic carbon with vinyl cyanide under single collision conditions implies that this molecule can be an important reaction intermediate in combustion flames and also in extraterrestrial environments (cold molecular clouds, circumstellar envelopes of carbon stars) which could lead to the formation of cyano benzene (C6H5CN) upon reaction with a propargyl radical.

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure.

The study was conducted in a Chinese population with occupational or environmental exposures to polycyclic aromatic hydrocarbons (PAHs). A total of 106 subjects were recruited from coke-oven workers (workers), residents in a metropolitan area (residents) and suburban gardeners (gardeners). All subjects were monitored twice for their personal exposures to PAHs. The biological samples were collected for measurements of 1-hydroxypyrene (1-OHP) and cotinine in urine. The geometric means of personal exposure levels of pyrene, benz(a)anthracene (BaA) and benzo(a)pyrene (BaP) in workers were 1.470, 0.978 and 0.805 microg m-3, respectively. The corresponding levels in residents were 0.050, 0.034 and 0.025 microg m-3; and those in gardeners were 0.011, 0.020 and 0.008 microg m-3, respectively. The conjugate of 1-OHP with glucuronide (1-OHP-G) is the predominant form of pyrene metabolite in urine and it showed strong associations with exposures not only to pyrene, but also to BaA, BaP and total PAHs. Most importantly, a significant difference in 1-OHP-G was even detected between the subgroups with exposures to BaP at < 0.010 and > 0.010 but < 0.020 microg m-3, suggesting that 1-OHP-G is a good marker that can be used for the risk assessment of BaP exposure at levels currently encountered in ambient air. Furthermore, multiple regression analyses of 1-OHP-G on PAHs exposure indicated that cigarette smoke was a major confounding factor and should be considered and adjusted for while using 1-OHP to estimate PAHs exposure.

Reaction of cyanoacetylene HCCCN(X 1Sigma+) with ground-state carbon atoms C(3P) in cold molecular clouds.

The reaction of the simplest cyanopolyyne, cyanoacetylene [HCCCN(X (1)Sigma(+))], with ground-state atomic carbon C((3)P) is investigated theoretically to explore the probable routes for the depletion of the famed interstellar molecule HCCCN, and the formation of carbon-nitrogen-bearing species in extraterrestrial environments particularly of ultralow temperature. Six collision complexes (c1-c6) without entrance barrier as a result of the carbon atom addition to the pi systems of HCCCN are located. The optimized geometries and harmonic frequencies of the intermediates, transition states, and products along the isomerization and dissociation pathways of each collision complex are obtained by utilizing the unrestricted B3YLP6-311G(d,p) level of theory, and the corresponding CCSD(T)/cc-pVTZ energies are calculated. Subsequently, with the facilitation of Rice-Ramsperger-Kassel-Marcus (RRKM) and variational RRKM rate constants at collision energy of 0-10 kcal/mol, the most probable paths for the titled reaction are determined, and the product yields are estimated. Five collision complexes (c1-c3, c5, and c6) are predicted to give the same products, a chained CCCCN (p2)+H, via the linear and most stable intermediate, HCCCCN (i2), while collision complex c4 is likely to dissociate back to C+HCCCN. The study suggests that this class of reaction is an important route to the destruction of cyanoacetylene and cyanopolyynes in general, and to the synthesis of linear carbon-chained nitriles at the temperature as low as 10 K to be incorporated in future chemical models of interstellar clouds.

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles.

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.

Dose-related effects of metoprolol on heart rate and pharmacokinetics in heart failure.

BACKGROUND: The pharmacokinetics and pharmacodynamics of immediate-release (IR) metoprolol, 50 mg 3 times daily, were compared with those of different doses of controlled-release/extended-release metoprolol (CR/XL) given once daily. METHODS AND RESULTS: Fifteen patients with chronic heart failure were randomized to a 3-way crossover study to receive metoprolol IR 50 mg 3 times daily, CR/XL 100 mg once daily, and CR/XL 200 mg once daily for 7 days. On the seventh day of each treatment, serial plasma samples were drawn and standardized exercise tests and a 24-hour Holter recording were performed. Metoprolol IR 50 mg produced peak plasma levels comparable to those observed for CR/XL 200 mg (285 v 263 nmol/L). The difference in mean 24-hour heart rate between CR/XL 100 mg and IR 50 mg was 1.0 bpm (95% confidence interval [CI]), -2.9 to 4.9; NS) compared with -3.8 bpm (95% CI, -7.6 to -0.04; P = .048) between CR/XL 200 mg and IR 50 mg. Submaximal exercise heart rate was lower for patients receiving CR/XL 200 mg than those receiving IR 50 mg. No difference in tolerance or exercise performance was observed between treatment regimens. CONCLUSIONS: Peak plasma levels produced by metoprolol 200 mg CR/XL were similar to those of 50 mg IR. Metoprolol CR/XL 200 mg was associated with a more pronounced suppression of heart rate than metoprolol IR 50 mg. It is suggested that patients can safely be switched from multiple dosing of metoprolol IR 50 mg to a once-daily dose of metoprolol CR/XL.

Early changes in longitudinal performance predict future improvement in global left ventricular function during long term beta adrenergic blockade.

OBJECTIVE: Contraction of longitudinal and subendocardial myocardial muscle fibres is reflected in descent of the atrioventricular (AV) plane. The aim was therefore to determine whether beta blocker treatment with prolongation of diastole might result in improved function as reflected by AV plane movements in patients with chronic heart failure. DESIGN: Double blind, randomised, placebo controlled and open intervention study. SETTING: University hospital. PATIENTS: Patients with congestive heart failure: placebo controlled (n = 26) and an open protocol (n = 15). INTERVENTIONS: 12 months of metoprolol treatment. MAIN OUTCOME MEASURES: Short axis and long axis echocardiography, invasive haemodynamics, radionuclide angiography. RESULTS: Recovery of systolic and diastolic function during metoprolol treatment was reflected by early changes in mean (SD) AV plane amplitude, from 5.3 (2.0)% to 7.1 (3.2)% and 7.8 (3. 1)% (at 3 and 12 months, respectively; p < 0.05). In a multivariate analysis, only the change in AV plane amplitude by three months was independently associated with improvement in pulmonary capillary wedge pressure by six months (r = 0.80, p = 0.017). Change in AV plane amplitude by three months was also a better predictor of improvement in ejection fraction by 12 months (r = 0.78, p < 0.001) than changes in radionuclide ejection fraction by three months (r = 0.34, p = 0.049). CONCLUSIONS: Improvement in longitudinal contraction was closely associated with a decrease in left ventricular filling pressure during metoprolol treatment. This association was stronger than changes in short axis performance or radionuclide ejection fraction, emphasising the importance of AV plane motion for left ventricular filling and systolic performance in patients with heart failure.

Both (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA- mutations at Codon 61 of c-H-ras.

We have determined the tumor-initiating activity of (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin.

Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide-DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene.

We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (+/-)anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers. Given the far reaching ramifications of such conclusions for cancer etiology, a legitimate question is raised regarding the reliability of using the UvrABC incision method for quantifying and determining the sequence-dependency of adduct formation. Is the higher frequency of UvrABC cutting at methylated versus unmethylated CpG sites due to the preference of the nuclease for cutting at those sites or due to the preferential formation of BPDE adducts at those sites? In order to distinguish between these two possibilities, we have analyzed the kinetics of UvrABC incision at BPDE adducts formed at either methylated CpG sites versus other sequences, or unmethylated CpG sites versus other sequences in exon 5 of the p53 gene. We have found that the UvrABC cutting kinetics are identical for both cases. On the basis of these results we conclude that under proper cutting conditions, UvrABC nuclease reacts with and incises with equal efficiency, BPDE adducts formed at methylated or unmethylated CpG sites as well as other sequences, and that the extent of UvrABC incision accurately reflects the extent of BPDE-DNA adduct formation. These conclusions were further supported by results obtained using a DNA synthesis blockage assay.

Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots.

The major mutational hot spots in human cancers occur at CpG sequences in the p53 gene. It is generally presumed that the majority of mutations at these sites result from the endogenous deamination of methylated cytosine. Using a UvrABC incision method, we have found that cytosine methylation greatly enhances guanine alkylation at all CpG sites in the p53 gene by a variety of carcinogens, including benzo(a)pyrene diol epoxide, benzo(g)chrysene diol epoxide, aflatoxin B1 8,9-epoxide, and N-acetoxy-2-acetylaminofluorene. These findings suggest that mutational hot spots at methylated CpG sequences in the p53 gene may be a consequence of preferential carcinogen binding at these sites.

Cyclobutane thymine dimers with a disrupted phosphodiester bond are refractory to T4 endonuclease V digestion but have increased sensitivity to UvrABC nuclease.

UV irradiation induces the dimerization of synthetic single-stranded, 80-mer oligonucleotides with self-complementary, alternating purine-pyrimidine sequences, and terminal 5'- and 3'-thymines; this process can be reversed by photoreactivation. The UV-induced 160-mers are sensitive to digestion by the restriction enzyme SnaBI, but monomers are insensitive to digestion, indicating that UV irradiation stabilizes the formation of double-stranded DNA. These results suggest that UV irradiation of these 80-mer oligonucleotide substrates induces the formation of a novel cyclobutane thymine dimer which lacks an intradimer phosphodiester bond (CPD*). This CPD*, linking the terminal thymines of two separate 80-mer molecules, is formed in a double-stranded DNA region created by self-annealing and intermolecular hybridization of the two 80-mer strands. We have found that these UV-induced CPD* in 160-mers are sensitive to cleavage by the nucleotide excision enzyme complex UvrABC nuclease, but resistant to cleavage by the cyclobutane pyrimidine dimer-specific enzyme T4 endonuclease V. However, pretreatment of the 160-mers with ligase reverses their sensitivity to these two enzymes, significantly reducing their susceptibility to cleavage by UvrABC nuclease but dramatically increasing their susceptibility to cleavage by T4 endonuclease. The biological significance of these findings is discussed.

Rate of incision of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts by UvrABC nuclease is adduct- and sequence-specific: comparison of the rates of UvrABC nuclease incision and protein-DNA complex formation.

The UvrABC nuclease, the nucleotide excision repair complex from Escherichia coli, is able to incise a variety of types of DNA damage and the repair efficiency of this enzyme complex appears to be influenced by the structure of the damage and the sequence context within which the damage is positioned. In order to better establish these relationships, we have constructed two DNA sequences each containing a site-specifically positioned N-2-aminofluorene (AF) or N-acetyl-2-aminofluorene (AAF) adduct and have determined both the kinetics of UvrABC nuclease incision and the kinetics of UvrABC nuclease-substrate complex formation. It is well established that these two adducts induce very different structures in the DNA and that these structures also depend on the sequence context. We have found that the rate of incision of both AAF- and AF-DNA adducts is significantly faster when they are positioned in the mutation hotspot NarI sequence (5-GGCG*CC-3') than when located in a normal or non-NarI sequence (5'-GATG*ATA-3') and that the rate of incision for AAF-DNA adducts is faster that for AF adducts in both sequences. Most siginificantly, we find that the rate of UvrB and UvrBC-substrate complex formation correlates with the rate of UvrABC nuclease incision.

PCR-based approaches to adduct analysis.

Ligation-mediated polymerase chain reaction (LMPCR) is a PCR-based method for the detection of DNA adducts at individual nucleotide positions in mammalian genes. Adduct-specific enzymes, such as T4 endonuclease V, various base excision repair enzymes, UvrABC nuclease, and chemical cleavage techniques can be used to convert the adducts into DNA strand breaks. The positions of these breaks are then detected by LMPCR. This method has been used primarily to map the distribution of UV-induced DNA lesions and adducts of polycyclic aromatic hydrocarbons. The number and diversity of mutations in the p53 mutation database provides indirect evidence that environmental mutagens may be involved in human carcinogenesis. We hypothesize that there is a limited involvement of selection for specific mutations in the central domain of the p53 protein, and that the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations, of certain cancers. This concept has been validated by experiments with sunlight and the cigarette smoke component benzo[a]pyrene representing the polycyclic aromatic hydrocarbon class of carcinogens. The damage and repair data obtained for these mutagens can predict certain parameters of the mutational spectra of human non-melanoma skin cancers and lung cancers from smokers. Future studies with suspected mutagens may help to implicate causative agents involved in other cancers, where the exact carcinogen has not yet been identified but an environmental factor is suspected.

Detection of an Fe2+-protoporphyrin-IX intermediate during aspirin-treated prostaglandin H2 synthase II catalysis of arachidonic acid to 15-HETE.

Spectral intermediates associated with the dioxygenase and peroxidase activities of prostaglandin H2 (PGH2) synthase I and II were monitored by stopped-flow spectrometry. During reactions of PGH2 synthase I with arachidonic acid (AA) and ethyl hydrogen peroxide (EtOOH), compound I (Fe5+; formally (protoporphyrin-IX) x +Fe4+=O) and compound II (Fe4+; formally (protoporphyrin-IX)Fe4+=O) were detected. These intermediates were observed sooner with EtOOH (within 50 ms) than with AA (within 200 ms). Compound I and compound II were found to be kinetically competent with respect to AA-dependent O2 uptake. These findings are consistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. During reactions with PGH2 synthase II with AA, compound I and compound II were again observed within 200 ms and were kinetically competent to participate in dioxygenation. However, during reactions of PGH2 synthase II with EtOOH, compound I and compound II were detected much later (after 10 s). These findings would be inconsistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. When aspirin-treated PGH2 synthase II was reacted with EtOOH, a normal peroxidase cycle occurred with compound I and compound II formation occurring over 10 s. However, when aspirin-treated PGH2 synthase II was reacted with AA, a unique spectral intermediate with lambda(max) at 446 nm was detected within 3 ms and was strikingly similar to ferrous (Fe2+) protoporphyrin-IX. Aspirin-treated PGH2 synthase II was found to produce 15-HETE, and the appearance of the Fe2+ intermediate (within 3 ms) indicated that it was kinetically competent to participate in the 15-dioxygenation event. The detection of this Fe2+ intermediate and the slow formation of compound I and compound II observed with EtOOH in PGH2 synthase II suggest that peroxidative cleavage is not the initiating event in dioxygenation. Instead, it is proposed that the reduction of Fe3+ in heme to Fe2+ oxidizes a peroxide to yield an initiating peroxy radical. Since it is unlikely that 11- and 15-dioxygenation occurs via different mechanisms, our findings question mechanisms of catalysis in both PGH2 synthases.

Cytosine methylation determines hot spots of DNA damage in the human P53 gene.

In the P53 tumor suppressor gene, a remarkably large number of somatic mutations are found at methylated CpG dinucleotides. We have previously mapped the distribution of (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts along the human P53 gene [Denissenko, M. F., Pao, A., Tang, M.-s. & Pfeifer, G. P. (1996) Science 274, 430-432]. Strong and selective formation of adducts occurred at guanines in CpG sequences of codons 157, 248, and 273, which are the major mutational hot spots in lung cancer. Chromatin structure was not involved in preferential modification of these sites by BPDE. To investigate other possible mechanisms underlying the selectivity of BPDE binding, we have mapped the adducts in plasmid DNA containing genomic P53 sequences. The adduct profile obtained was different from that in genomic DNA. However, when cytosines at CpG sequences were converted to 5-methylcytosines by the CpG-specific methylase SssI and the DNA was subsequently treated with BPDE, adduct hot spots were created which were similar to those seen in genomic DNA where all CpGs are methylated. A strong positive effect of 5-methylcytosine on BPDE adduct formation at CpG sites was also documented with sequences of the PGK1 gene derived from an active or inactive human X chromosome and having differential methylation patterns. These results show that methylated CpG dinucleotides, in addition to being an endogenous promutagenic factor, may represent a preferential target for exogenous chemical carcinogens. The data open new avenues concerning the reasons that the majority of mutational hot spots in human genes are at CpGs.