A Highly Verified Assay for KRAS Mutation Detection in Tissue and Plasma of Lung, Colorectal, and Pancreatic Cancer.

CONTEXT.­: KRAS Mutation Test v2 is used for the qualitative detection and identification of 28 mutations in exons 2, 3, and 4 of the human KRAS gene. OBJECTIVE.­: To verify the performance of KRAS Mutation Test v2 and to evaluate its accuracy by correlation with a next-generation sequencing method on Illumina MiSeq. DESIGN.­: In this study, we used formalin-fixed, paraffin-embedded tissue and plasma specimens from non-small cell lung cancer, colorectal cancer, and pancreatic cancer patients. Results of specificity, precision, analytical sensitivity, and accuracy as compared with a MiSeq method are reported. RESULTS.­: The KRAS Mutation Test v2 demonstrated exquisite sensitivity and specificity and broad coverage of KRAS mutations. Precision was 100% (108 of 108) across all samples, operators, and instruments for formalin-fixed, paraffin-embedded tissue and 99.8% (615 of 616) for plasma. Analytical sensitivity was high with detection of 1% mutant sequence in formalin-fixed, paraffin-embedded tissue samples and as low as 25 mutant sequence copies/mL for plasma samples. The test also showed high overall concordance for formalin-fixed, paraffin-embedded tumor tissue as well as for plasma specimens when compared with MiSeq sequencing results. CONCLUSIONS.­: The KRAS Mutation Test v2 is a highly robust, reproducible, and sensitive test for the qualitative detection of 28 mutations in exons 2, 3, and 4 of the KRAS gene in both solid (tissue) and liquid (plasma) biopsies from colorectal cancer, non-small cell lung cancer, and pancreatic cancer, and is a convenient option for KRAS mutation testing.

Paraoxonase 1 (PON1) C/T-108 association with longitudinal mean arterial blood pressure.

BACKGROUND: Blood pressure is a complex quantitative trait with a strong genetic component. In this study, we leveraged the Veterans Affairs electronic medical record system to explore the relationship between Paraoxonase 1 (PON1)-108 C/T (rs705379) and mean arterial blood pressure (MAP). METHODS: Outpatient blood pressure data over an approximate 8-year period was collected from the Veterans Affairs Hypertensive Cohort (N = 1,302). Association between genotype and longitudinal MAP was further explored using a random effects model controlling for age, ancestry, renal function, and other determinants of blood pressure. To control for population stratification, principal component groupings based on ancestry informative markers in this dataset were included as covariates (in addition to self-identified ancestry). Data from the African American Study of Kidney Disease and Hypertension (AASK, N = 857) was used to confirm significant findings in an independent cohort. RESULTS: There was a significant interaction between PON1-108 C/T genotype and follow-up age group. At a younger age (<50 years), there was an estimated 2.53 mm Hg (95% confidence interval: 1.06, 4.00) increase in MAP with each additional C-allele. At the older age groups, there were no significant associations between PON1-108 C/T genotype and MAP. Using data from the AASK trial, the C-allele at PON1-108 C/T was significantly associated with a higher MAP (P = 0.005) but only among younger participants (<54 years). CONCLUSIONS: The PON1-108 polymorphism may be associated with MAP in an age-dependent manner.

Analytical performance of a PCR assay for the detection of KRAS mutations (codons 12/13 and 61) in formalin-fixed paraffin-embedded tissue samples of colorectal carcinoma.

KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe the performance of a TaqMelt polymerase chain reaction (PCR) assay-the cobas® KRAS Mutation Test-designed to detect 19 mutations in codons 12, 13, and 61. The limit of detection was determined using DNA blends from cell lines, plasmids, and formalin-fixed paraffin-embedded tissue specimens. Assay performance was compared to Sanger sequencing using a panel of 188 specimens. Discordant specimens were subjected to next generation pyrosequencing (454). Assay repeatability was assessed using a panel of six specimens. A >95% correct mutation call rate was obtained in all specimen types with ~5% mutant alleles at DNA inputs of 0.8-6.3 ng per PCR reaction; 100% detection rate was observed at the recommended DNA input of 50 ng. The positive percent agreement with Sanger was 97.5% (79/81) for codons 12/13 and 85.7% (6/7) for codon 61. Negative percent agreement was 94.4% (101/107) for codon 12/13 and 99.4% (180/181) for codon 61. Nine of 10 discordant specimens yielded 454 results consistent with the cobas® results. With repeated testing, the assay showed a correct call rate of 100% (192/192) for all operators, instruments, reagent lots, and days tested. The cobas® test detects KRAS mutations in codons 12, 13, and 61 at a limit of detection of <5%. The PCR assay was more sensitive and specific than Sanger sequencing, and performance was highly reproducible. Test performance was not influenced by various endogenous interfering substances or common gut microbes.

Pharmacogenetic associations of MMP9 and MMP12 variants with cardiovascular disease in patients with hypertension.

OBJECTIVES: MMP-9 and -12 function in tissue remodeling and may play roles in cardiovascular disease (CVD). We assessed associations of four MMP polymorphisms and three antihypertensive drugs with cardiovascular outcomes. METHODS: Hypertensives (n = 42,418) from a double-blind, randomized, clinical trial were randomized to chlorthalidone, amlodipine, lisinopril, or doxazosin treatment (mean follow up, 4.9 years). The primary outcome was coronary heart disease (CHD). Secondary outcomes included combined CHD, all CVD outcomes combined, stroke, heart failure (HF), and mortality. Genotype-treatment interactions were tested. RESULTS: There were 38,698 participants genotyped for at least one of the polymorphisms included here. For MMP9 R668Q (rs2274756), lower hazard ratios (HRs) were found for AA subjects for most outcomes when treated with chlorthalidone versus amlodipine (eg., CCHD: GG = 1.00, GA = 1.01, AA = 0.64; P = 0.038). For MMP9 R279Q (rs17576), modest pharmacogenetic findings were observed for combined CHD and the composite CVD outcome. For MMP12 N122S (rs652438), lower HRs were observed for CHD in subjects carrying at least one G allele and being treated with chlorthalidone versus lisinopril (CHD: AA = 1.07, AG = 0.80, GG = 0.49; P = 0.005). In the lisinopril-amlodipine comparison, higher HRs were observed for participants having at least one G allele at the MMP12 N122S locus (CHD: AA = 0.94, AG = 1.19, GG = 1.93; P = 0.041). For MMP12 -82A>G (rs2276109), no pharmacogenetic effect was found for the primary outcome, although lower HRs were observed for AA homozygotes in the chlorthalidone-amlodipine comparison for HF (P = 0.015). CONCLUSIONS: We observed interactions between antihypertensive drugs and MMP9 and MMP12 for CHD and composite CVD. The data suggest that these genes may provide useful clinical information with respect to treatment decisions.

ß(1)-adrenergic receptor polymorphisms and response to ß-blockade in the African-American study of kidney disease and hypertension (AASK).

BACKGROUND: This study focuses on the relationship between ß(1)-adrenergic receptor (ADRB1) polymorphisms and blood pressure response to the ß-blocker metoprolol among African Americans with early hypertensive nephrosclerosis. METHODS: Participants from the African-American Study of Kidney Disease and Hypertension (AASK) trial were genotyped for ADRB1 polymorphisms: Ser49Gly and Arg389Gly. Cox proportional hazards models were used to determine the relationship between ADRB1 polymorphisms and time to reach a mean arterial pressure (MAP) of ≤107 mm Hg in the first year after randomization, adjusted for other predictors of blood pressure response. RESULTS: In the Ser49Gly model, Ser49/Gly49 individuals were less responsive compared to Ser49/Ser49 only among the more obese (body mass index (BMI) ≥39 kg/m(2)) participants (P < 0.05 for genotype × BMI interaction). The hazard ratio (HR) with a BMI of 39 kg/m(2) was 0.68 (95% confidence interval (CI) 0.46-0.99). In the Arg389Gly model, participants with Arg389 were less likely to respond to metoprolol: HR: 0.68 (95% CI 0.50-0.93). In addition, women were less responsive to metoprolol compared to men: HR: 0.78 (95% CI 0.60-0.995). CONCLUSIONS: Ser49/Gly49 was predictive of blood pressure response to metoprolol only among more obese African Americans with early hypertensive nephrosclerosis. In contrast to other studies suggesting increased short-term responsiveness to ß-blockers with Arg389, Arg389 individuals were less responsive in this study analyzing blood pressure over a 1-year period. This may be partly explained by decreased agonist-promoted desensitization with Arg389. However, gender, physiological adaption to stress, interactions between genes and between genes and the environment, as well as study in other patient populations need to be considered.

Matrix metalloproteinase haplotypes associated with coronary artery aneurysm formation in patients with Kawasaki disease.

Aneurysms of the vascular wall represent a final common pathway for a number of inflammatory processes, including atherosclerosis and idiopathic vasculitis syndromes. Kawasaki disease (KD) is an acute, self-limited vasculitis in children and the leading cause of acquired coronary artery aneurysms. We sought to identify shared molecular mechanisms of aneurysm formation by genotyping eight polymorphisms in matrix metalloproteinase (MMP)-1, 3, 7, 12 and 13 in the gene cluster on Chr.11q22, whose gene products have been implicated in aneurysm formation or are known to have elastase activity. We genotyped 482 US-UK KD patients (aneurysm+: n=111, aneurysm-: n=371) and tested our findings in an independent cohort of 200 Japanese KD patients (aneurysm+: n=58, aneurysm-: n=142). Analysis of the five MMP genes identified modest trends in allele and genotype frequencies for MMP-3 rs3025058 (-/T) and haplotypes containing MMP-3 rs3025058 (-/T) and MMP-12 rs2276109 (A/G) (nominal P=2 to 4 × 10(-5)) that conferred increased risk of aneurysm formation in US-UK subjects. This finding was validated in Japanese subjects and suggests the importance of this locus in aneurysm formation in children with KD. The region encompassing these risk haplotypes is a prime candidate for resequencing to look for rare genetic variation that may influence aneurysm formation.

Progression of chronic kidney disease: Adrenergic genetic influence on glomerular filtration rate decline in hypertensive nephrosclerosis.

BACKGROUND: African-Americans are likely to develop hypertension and hypertensive nephrosclerosis. This grave prognosis, coupled with familial aggregation of end-stage renal disease (ESRD) in Blacks, prompts a search for genetic risk factors for ESRD. Recent evidence implicates a crucial role for the sympathetic nervous system in progressive renal disease. METHODS: We used the African-American Study of Kidney Disease to probe whether beta2-adrenergic receptor (ADRB2) predicts glomerular filtration rate (GFR) decline rate. A total of 580 participants were included. Baseline GFR was 51.2 +/- 0.5 ml/min/1.73 m2. Subjects were randomized in a 2 x 3 block design: to intensively lowered (MAP < or = 92 mm Hg) versus 'usual' (MAP = 102-107 mm Hg) blood pressure goal groups, and also divided by three randomized antihypertensive drugs (ramipril, metoprolol, or amlodipine). We scored 4 SNPs at the ADRB2 locus. RESULTS: Haplotypes at ADRB2 predicted chronic GFR decline rate, GFR declined more slowly in individuals with haplotype-1 (-804G-->173T-->16Gly-->27GIn), and faster in those who carried haplotype-3 (-804G-->173T-->16Arg-->27Gln). ADRB2 genotype interacted with antihypertensive drug class to influence GFR slope (p = 0.001-0.037). We extended our findings to an independent case/control sample of Black hypertensive ESRD, in which we found that variant Gly16Arg that tagged the GFR slope-determining ADRB2 haplotype also conferred risk for the ESRD trait in Blacks. CONCLUSIONS: The GFR decline/progression rate in hypertensive renal disease is controlled in part by genetic variation within the adrenergic pathway.

The VA Hypertension Primary Care Longitudinal Cohort: Electronic medical records in the post-genomic era.

The Veterans Affairs Hypertension Primary Care Longitudinal Cohort (VAHC) was initiated in 2003 as a pilot study designed to link the VA electronic medical record system with individual genetic data. Between June 2003 and December 2004, 1,527 hypertensive participants were recruited. Protected health information (PHI) was extracted from the regional VA data warehouse. Differences between the clinic and mail recruits suggested that clinic recruitment resulted in an over-sampling of African Americans. A review of medical records in a random sample of study participants confirmed that the data warehouse accurately captured most selected diagnoses. Genomic DNA was acquired non-invasively from buccal cells in mouthwash; ~ 96.5 per cent of samples contained DNA suitable for genotyping, with an average DNA yield of 5.02 ± 0.12 micrograms, enough for several thousand genotypes. The coupling of detailed medical databases with genetic information has the potential to facilitate the genetic study of hypertension and other complex diseases.

CYP3A4 and CYP3A5 polymorphisms and blood pressure response to amlodipine among African-American men and women with early hypertensive renal disease.

PURPOSE: To explore the association between CYP3A4 and CYP3A5 gene polymorphisms and blood pressure response to amlodipine among participants from the African-American Study of Kidney Disease and Hypertension Trial randomized to amlodipine (n = 164). METHODS: Cox proportional hazards models were used to determine the risk of reaching a target mean arterial pressure (MAP) of < or =107 mm Hg by CYP3A4 (A-392G and T16090C) and CYP3A5 (A6986G) gene polymorphisms, stratified by MAP randomization group (low or usual) and controlling for other predictors for blood pressure response. RESULTS: Women randomized to a usual MAP goal with an A allele at CYP3A4 A-392G were more likely to reach a target MAP of 107 mm Hg. The adjusted hazard ratio (AA/AG compared to GG) with 95% confidence interval was 3.41 (1.20-9.64; p = 0.020). Among participants randomized to a lower MAP goal, those with the C allele at CYP3A4 T16090C were more likely to reach target MAP: The adjusted hazard ratio was 2.04 (1.17-3.56; p = 0.010). After adjustment for multiple testing using a threshold significance level of p = 0.016, only the CYP3A4 T16090C SNP remained significant. CYP3A5 A6986G was not associated with blood pressure response. CONCLUSIONS: Our findings suggest that blood pressure response to amlodipine among high-risk African-Americans appears to be determined by CYP3A4 genotypes, and sex specificity may be an important consideration. Clinical applications of CYP3A4 genotype testing for individualized treatment regimens warrant further study.

Adrenergic beta-1 receptor genetic variation predicts longitudinal rate of GFR decline in hypertensive nephrosclerosis.

BACKGROUND: End-stage renal disease (ESRD) due to hypertension is common and displays familial aggregation in African Americans, suggesting genetic risk factors, including adrenergic activity alterations which are noted in both hypertension and ESRD. METHODS: We analysed 554 hypertensive nephrosclerosis participants (without clinically significant proteinuria) from the longitudinal National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) African American Study of Kidney Disease and Hypertension (AASK) cohort to determine whether decline in glomerular filtration rate (GFR) over approximately 3.8 years was predicted by common genetic variation within the adrenergic beta-1 (ADRB1) receptor at non-synonymous positions Ser49Gly and Arg389Gly. RESULTS: The polymorphism at Ser49Gly (though not Arg389Gly, in only partial linkage disequilibrium at r(2) = 0.18) predicted the chronic rate of GFR decline, with minimal decline in Gly(49)/Gly(49) (minor allele) homozygotes compared to Ser(49) carriers; concordant results were observed for haplotypes and diploid haplotype pairs at the locus. An independent replication study in 1244 subjects from the San Diego Veterans Affairs Hypertension Cohort confirmed that Gly(49)/Gly(49) homozygotes displayed the least rapid decline of eGFR over approximately 3.6 years. CONCLUSION: We conclude that GFR decline rate in hypertensive renal disease is controlled in part by genetic variation within the adrenergic pathway, particularly at ADRB1. The results suggest novel strategies to approach the role of the adrenergic system in the risk and treatment of progressive renal disease.

G-protein-coupled receptor kinase 4 polymorphisms and blood pressure response to metoprolol among African Americans: sex-specificity and interactions.

BACKGROUND: African Americans have a disproportionate burden of hypertension and comorbid disease. Pharmacogenetic markers of blood pressure response have yet to be defined clearly. This study explores the association between G-protein-coupled receptor kinase type 4 (GRK4) variants and blood pressure response to metoprolol among African Americans with early hypertensive nephrosclerosis. METHODS: Participants from the African American Study of Kidney Disease and Hypertension (AASK) trial were genotyped at three GRK4 polymorphisms: R65L, A142V, and A486V. A Cox proportional hazards model, stratified by gender, was used to determine the relationship between GRK4 variants and time to reach a mean arterial pressure (MAP) of 107 mm Hg, adjusted for other predictors of blood pressure response. Potential interactions between the three polymorphisms were explored by analyzing the effects of gene haplotypes and by stratifying the analysis by neighboring sites. RESULTS: The hazard ratio with 95% confidence interval by A142V among men randomized to a usual MAP (102-107 mm Hg) was 1.54 (1.11-2.44; P = 0.0009). The hazard ratio by A142V with R65/L65 or L65/L65 was 2.14 (1.35-3.39; P = 0.001). Haplotype analyses were consistent but inconclusive. There was no association between A142V and blood pressure response among women. CONCLUSIONS: Results suggest a sex-specific relationship between GRK4 A142V and blood pressure response among African-American men with early hypertensive nephrosclerosis. Men with a GRK4 A142 were less responsive to metoprolol if they had a GRK4 L65 variant. The effect of GRK4 variants and blood pressure response to metoprolol should be studied in larger clinical trials.

A meta-analysis of candidate gene polymorphisms and ischemic stroke in 6 study populations: association of lymphotoxin-alpha in nonhypertensive patients.

BACKGROUND AND PURPOSE: Ischemic stroke is a multifactorial disease with a strong genetic component. Pathways, including lipid metabolism, systemic chronic inflammation, coagulation, blood pressure regulation, and cellular adhesion, have been implicated in stroke pathophysiology, and candidate gene polymorphisms in these pathways have been proposed as genetic risk factors. METHODS: We genotyped 105 simple deletions and single nucleotide polymorphisms from 64 candidate genes in 3550 patients and 6560 control subjects from 6 case-control association studies conducted in the United States, Europe, and China. Genotyping was performed using the same immobilized probe typing system and meta-analyses were based on summary logistic regressions for each study. The primary analyses were fixed-effects meta-analyses adjusting for age and sex with additive, dominant, and recessive models of inheritance. RESULTS: Although 7 polymorphisms showed a nominal additive association, none remained statistically significant after adjustment for multiple comparisons. In contrast, after stratification for hypertension, 2 lymphotoxin-alpha polymorphisms, which are in strong linkage disequilibrium, were significantly associated among nonhypertensive individuals: LTA 252A>G (additive model; OR, 1.41 with 95% CI, 1.20 to 1.65; P=0.00002) and LTA 26Thr>Asn (OR, 1.19 with 95% CI, 1.06 to 1.34; P=0.003). LTA 252A>G remained significant after adjustment for multiple testing using either the false discovery rate or by permutation testing. The 2 single nucleotide polymorphisms showed no association in hypertensive subjects (eg, LTA 252A>G, OR, 0.93; 95% CI, 0.84 to 1.03; P=0.17). CONCLUSIONS: These observations may indicate an important role of LTA-mediated inflammatory processes in the pathogenesis of ischemic stroke.

Polymorphisms of the phosphodiesterase 4D, cAMP-specific (PDE4D) gene and risk of ischemic stroke: a prospective, nested case-control evaluation.

BACKGROUND AND PURPOSE: In an Icelandic population, gene variants of the phosphodiesterase 4D, cAMP-specific (PDE4D) gene were reported to be risk predictors for ischemic stroke. Case-control studies in other populations have yielded mixed evidence for association. A recent analysis in a prospective, non-Icelandic study found an association with stroke after stratification by hypertension. METHODS: We evaluated nine PDE4D single nucleotide polymorphisms (SNPs) among 259 incident ischemic stroke cases and 259 controls were matched on age and smoking status and length of follow up since randomization, all drawn from initially healthy white males within the Physicians' Health Study cohort who were prospectively followed for first-ever stroke events. RESULTS: Genotype and allele distributions were similar between cases and controls. Results from single-marker conditional logistic regression analysis adjusting for traditional stroke risk factors showed significant association of SNP56 with risk of ischemic stroke (recessive odds ratio [OR], 2.26; 95% confidence interval [CI], 1.11 to 4.61; P=0.03). Among the participants without baseline hypertension, SNP42 (additive OR, 1.68; 95% CI, 0.99 to 2.86, P=0.06), SNP45 (dominant odds ratio, 2.24; 95% CI, 1.00 to 5.00, P=0.05), and SNP56 (additive odds ratio, 1.77; 95% CI, 1.02 to 3.10, P=0.04) showed modest association with increased risk of ischemic stroke. CONCLUSIONS: We found modest associations between several PDE4D gene polymorphisms and risk of incident ischemic stroke in men without baseline hypertension in this prospective, non-Icelandic study. Although of borderline statistical significance, the direction and magnitude of the effect for SNP42 parallels that observed in a recent study evaluating women from an independent, nested case-control study.

Association of phosphodiesterase 4D polymorphisms with ischemic stroke in a US population stratified by hypertension status.

BACKGROUND AND PURPOSE: Phosphodiesterase 4D (PDE4D) underlies the STRK1 linkage peak for stroke on chromosome 5q12 identified in Iceland. We tested association of 13 single-nucleotide polymorphisms (SNPs) and 1 microsatellite in a nested case-control sample of elderly white women (>65 years of age) from the Study of Osteoporotic Fractures (SOF) in the United States. METHODS: The genotypes of 248 women who experienced an incident ischemic stroke during an average of 5.4 years of follow-up were compared with 560 controls. RESULTS: Marginal associations with stroke (P<0.10) were found for 3 polymorphisms. Stratification of the population by hypertension markedly strengthened the association. SNPs 9 (hazard ratio [HR], 0.48; 95% CI, 0.26 to 0.91), 42 (HR, 1.73; 95% CI, 1.10 to 2.70), 219 (HR, 1.73; 95% CI, 1.13 to 2.64), and 220 (HR, 1.56; 95% CI, 1.05 to 2.32) showed significant association with stroke (P<0.05) under a dominant model in subjects without hypertension at baseline, and SNP 175 was significantly associated with stroke under an additive model (HR, 0.76; 95% CI, 0.59 to 0.98) in subjects with hypertension. Furthermore, the microsatellite AC008818-1 showed association with stroke only in the nonhypertensive subjects. Based on results in Iceland, specific haplotypes were tested in SOF, and stratification by hypertension also affected these association results. CONCLUSIONS: These data are consistent with an association of the PDE4D gene with stroke in a non-Icelandic sample and suggest an effect of hypertension status.

Paraoxonase activity, but not haplotype utilizing the linkage disequilibrium structure, predicts vascular disease.

OBJECTIVE: The effects of paraoxonase (PON1) activity and of genetic variation in the PON1 promoter and coding region on carotid artery disease (CAAD) were investigated. METHODS AND RESULTS: We identified functional promoter polymorphisms and examined their effects in a cohort with and without CAAD. We used the full sequences in 23 white subjects to determine the linkage disequilibrium (LD) structure of the PON1 region and to direct the grouping of haplotypes for disease association testing. There are several discrete regions of the PON1 gene with strong local LD, but the useful levels of LD do not extend across the entire gene. Indeed, PON1-162/-108/55/192 haplotype did not predict additional variation in PON1 activities compared with the 4 genotypes separately. PON1 hydrolysis activity predicted CAAD status, but this was not attributable to the promoter or coding region polymorphisms or haplotype or to the effects of smoking or statin use on PON1 activity. CONCLUSIONS: PON1 does not have LD across the gene, and use of haplotypes in association studies should consider the LD structure. PON1 activity predicts CAAD, yet 4 functional polymorphisms do not. Additional investigations of genetic and environmental factors that influence PON1 activity as a risk factor for vascular disease are warranted.

Vitamin C and E intake is associated with increased paraoxonase activity.

OBJECTIVE: Paraoxonase (PON1), an esterase physically associated with high density lipoprotein, has been shown to inhibit atherogenic low density lipoprotein and high density lipoprotein oxidation. PON1 activity appears to be primarily under genetic control with some environmental modification and is a predictor of vascular disease. Vitamins C and E, dietary antioxidants, scavenge free-oxygen radical products that may depress PON1 activity. Therefore, we evaluated the relationship between dietary vitamin C and E intake and PON1 activity. METHODS AND RESULTS: The vitamin C and E intakes of male white subjects (n=189) were estimated by using a standardized food frequency survey. With covariates, vitamin C or E intakes were found to be significant positive predictors of PON1 activity for the hydrolysis of paraoxon and diazoxon with the use of linear regression. Smoking and use of statins were independent predictors of PON1 activity. CONCLUSIONS: PON1 activity, which is primarily genotype dependent, varies with antioxidant vitamins, cigarette smoking, and statin drug use. Because PON1 activity is a better predictor of vascular disease than is the currently described genetic variation in PON1, further studies of the environmental influences on PON1 activity and additional PON1 genetic variants are warranted.

Treatment of COS-7 cells with proteasome inhibitors or gamma-interferon reduces the increase in caspase 3 activity associated with staurosporine-induced apoptosis.

Proteasomes play a major role in intracellular protein degradation and have been implicated in apoptosis. In this study we have investigated proteasome activity and the effects of inhibition of proteasomes or modulation of proteasome complexes on staurosporine-induced apoptosis in COS-7 cells. Staurosporine treatment of COS-7 cells had little direct effect on proteasome activity and did not cause dissociation of 26S proteasomes. There was also no major redistribution of proteasomes accompanying apoptosis in COS-7 cells. However, when the cells were pretreated with proteasome inhibitors, both the caspase 3 activity of the cells and the percentage of apoptotic cells measured by the TUNEL assay were reduced compared to staurosporine-treated cells, which had no inhibitor added. Proteasome inhibitors were also found to reduce the activation of caspase 3 in living cells which was assayed using a FRET-based method. However, proteasome inhibitors did not prevent some of the morphological changes associated with staurosporine-induced apoptosis. Pretreatment of cells with gamma-interferon, which increases immunoproteasomes and PA28 complexes and reduces 26S proteasome levels, had an antiapoptotic effect. These results are consistent with a role for 26S proteasomes in regulating the activation of caspase 3 through the degradation of key regulatory proteins.