BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent ki-nases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF SKP2 (SKP1/Cul1/F-box), the E3 ubiq-uitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G 1 arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2 V617F , and TEL-PDGFR, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic ty-rosine kinases. Mice that received transplants of BCR-ABL-infected SKP2 / marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2 / marrow. SKP2 / leukemic cells demonstrated higher levels of nuclear p27 than SKP2 / counterparts, suggesting that the attenu-ation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF SKP2 may be therapeutically useful. (Blood.

Flavin-containing monooxygenases (FMOs) are important for the disposition of many therapeutics, environmental toxicants, and nutrients. FMO3, the major adult hepatic FMO enzyme, exhibits significant interindividual variation. Eighteen FMO3 single-nucleotide polymorphism (SNP) frequencies were determined in 202 Hispanics (Mexican descent), 201 African Americans, and 200 non-Latino whites. Using expressed recombinant enzyme with methimazole, trimethylamine, sulindac, and ethylenethiourea, the novel structural variants FMO3 E24D and K416N were shown to cause modest changes in catalytic efficiency, whereas a third novel variant, FMO3 N61K, was essentially devoid of activity. The latter variant was present at an allelic frequency of 5.2% in non-Latino whites and 3.5% in African Americans, but it was absent in Hispanics. Inferring haplotypes using PHASE, version 2.1, the greatest haplotype diversity was observed in African Americans followed by nonLatino whites and Hispanics. Haplotype 2A and 2B, consisting of a hypermorphic promoter SNP cluster (-2650CϾG, -2543TϾA, and -2177GϾC) in linkage with synonymous structural variants was inferred at a frequency of 27% in the Hispanic population, but only 5% in non-Latino whites and African Americans. This same promoter SNP cluster in linkage with one or more hypomorphic structural variant also was inferred in multiple haplotypes at a total frequency of 5.6% in the AfricanAmerican study group but less than 1% in the other two groups. The sum frequencies of the hypomorphic haplotypes H3 [15,167GϾA (E158K)], H5B [-2650CϾG, 15,167GϾA (E158K), 21,375CϾT (N285N), 21,443AϾG (E308G)], and H6 [15,167GϾA (E158K), 21,375CϾT (N285N)] was 28% in Hispanics, 23% in non-Latino whites, and 24% in African Americans.The flavin-containing monooxygenases (FMOs; EC 1.14.13.8) are a family of microsomal enzymes that catalyze the NADPH-dependent N-and S-oxidation of a variety of therapeutics, environmental toxicants, carcinogens, and nutrients (Krueger and Williams, 2005). The FMO multigene family consists of a five-gene cluster at 1q24.3 (FMO1-4 and FMO6p), a second cluster of five genes at 1q24.2 (FMO7p-11p), and a single gene, FMO5, at 1q21.1, encoding a total of five active proteins in the human (Hernandez et al., 2004).The most recent common precursor for all placental mammals is predicted to have already had a cluster containing FMO1-6P and a separate FMO5 locus that arose from duplication of an ancestral gene approximately 210 to 275 million years ago (Hernandez et al., 2004). Despite the antiquity of the FMO gene family, a sequence comparison among modern FMO enzymes reveals 76 to 86% sequence identity between orthologous proteins, suggesting these genes have been highly conserved.The individual FMO enzymes exhibit broad but distinct substrate specificities (Krueger and Williams, 2005) as well as species-, sex-, tissue-, and age-dependent differences in expression patterns (for review, see Hines, 2006). In the human, FMO3 is the predominant adult hepatic enzyme with a specific conten...

Tamoxifen is utilized in breast cancer therapy and in chemoprevention. Tamoxifen may enhance risk for other neoplasias, especially endometrial cancer. The risk:benefit depends on the rate of metabolic activation versus detoxication. Cytochrome P450-dependent alpha-hydroxylation, followed by sulfonation, represents a metabolic activation pathway, producing products capable of covalent DNA adduction. In contrast, tamoxifen N-oxygenation represents a detoxication pathway, with the caveat that N-oxides can be reduced back to the parent amines. The N-oxygenation pathway will be the focus for this review. Dr. David Kupfer pioneered studies on cytochrome P450 and flavin-containing monooxygenase (FMO) tamoxifen metabolism. We collaborated with Dr. Kupfer's laboratory and recently determined that the low level of tamoxifen N-oxide production in human liver microsomes may be explained by the kinetics of FMO1 versus FMO3.

The significance of active vs. inactive flavin-containing monooxygenase 2 (FMO2) for human drug and xenobiotic metabolism and sensitivity is unknown, but the underlying ethnic polymorphism is well documented. We used quantitative real-time PCR to measure message levels of Fmo1, Fmo2, Fmo3 and Fmo5 in lung and liver from eight strains of eight week old female mice to determine if a strain could be identified that predominately expressed Fmo2 in lung, recapitulating the human FMO expression profile and being the ideal strain for Fmo2 knockout studies. We also characterized enzyme activity of baculovirus expressed mouse Fmo1, Fmo2 and Fmo3 to identify a substrate or incubation conditions capable of discriminating Fmo2 from Fmo mixtures. Fmo transcript expression patterns were similar for all strains. In lung, 59% of total FMO message was Fmo2, but Fmo1 levels were also high, averaging 34%, whereas Fmo3 and Fmo5 levels were 2 and 5%, respectively. In liver, Fmo1, Fmo2, Fmo3 and Fmo5 contributed 16, 1, 7 and 76% respectively, of detected message. Peak activity varied by isoform and was pH-and substrate-dependent. Fmo3 oxidation of methyl ptolyl sulfide was negligible at pH 9.5, but Fmo3 oxidation of methimazole was comparable to Fmo1 and Fmo2. Heating microsomes at 50 °C for 10 min eliminated most Fmo1 and Fmo3 activity, while 94% of Fmo2 activity remained. Measurement of activity in heated and unheated lung and liver microsomes verified relative transcript abundance. Our results show that dual Fmo1/2 knockouts will be required to model the human lung FMO profile.

Rationale Quantifying cellular proteins in ventricular myocytes (MCs) is challenging due to tissue heterogeneity and the variety of cell sizes in the heart. In post-weaning cardiac ontogeny, rod-shaped MCs make up the majority of the cardiac mass while remaining a minority of cardiac cells in number. Current biochemical analyses of cardiac proteins do not correlate well the content of MC-specific proteins to cell type or size in normally developing tissue. Objective To develop a new MC-specific large-particle fluorescent-activated cell sorting (LP-FACS) strategy for the purification of adult rod-shaped MCs. This approach is developed to enable growth-scaled measurements per-cell of the MC proteome and sarcomeric protein (i.e. myosin heavy chain (MyHC) and alpha-actin (α-actin)) content. Methods and Results Individual cardiac cells were isolated from 21-94 days old mice. An LP-FACS jet-in-air system with a 200-μm nozzle was defined by the first time to purify adult MCs. Cell-type specific immunophenotyping and sorting yielded ≥95% purity of adult MCs independently of cell morphology and size. This approach excluded other cell types and tissue contaminants from further analysis. MC proteome, MyHC and α-actin proteins were measured in linear biochemical assays normalized to cell numbers. Using the allometric coefficient α, we scaled the MC-specific rate of protein accumulation to growth post-weaning. MC-specific volumes (α=1.02) and global protein accumulation (α=0.94) were proportional (i.e. isometric) to body mass. In contrast, MyHC and α-actin accumulated at a much greater rate (i.e. hyperallometric) than body mass (α= 1.79 and 2.19 respectively) and MC volumes (α= 1.76 and 1.45 respectively). Conclusion Changes in MC proteome and cell volumes measured in LP-FACS purified MCs are proportional to body mass post-weaning. Oppositely, MyHC and α-actin are concentrated more rapidly than what would be expected from MC proteome accumulation, cell enlargement, or animal growth alone. LP-FACS provides a new standard for adult MC purification and an approach to scale the biochemical content of specific proteins or group of proteins per cell in enlarging MCs.