Cheol Koo Lee

Gene-expression profile of the ageing brain in mice

Ageing of the brain leads to impairments in cognitive and motor skills, and is the major risk factor for several common neurological disorders such as Alzheimer disease (AD) and Parkinson disease (PD). Recent studies suggest that normal brain ageing is associated with subtle morphological and functional alterations in specific neuronal circuits, as opposed to large-scale neuronal loss. In fact, ageing of the central nervous system in diverse mammalian species shares many features, such as atrophy of pyramidal neurons, synaptic atrophy, decrease of striatal dopamine receptors, accumulation of fluorescent pigments, cytoskeletal abnormalities, and reactive astrocytes and microglia. To provide the first global analysis of brain ageing at the molecular level, we used oligonucleotide arrays representing 6,347 genes to determine the gene-expression profile of the ageing neocortex and cerebellum in mice. Ageing resulted in a gene-expression profile indicative of an inflammatory response, oxidative stress and reduced neurotrophic support in both brain regions. At the transcriptional level, brain ageing in mice displays parallels with human neurodegenerative disorders. Caloric restriction, which retards the ageing process in mammals, selectively attenuated the age-associated induction of genes encoding inflammatory and stress responses.

Evidence for nucleosome depletion at active regulatory regions genome-wide

The identification of nuclease-hypersensitive sites in an active globin gene and in the 5′ regions of fruit fly heat shock genes first suggested that chromatin changes accompany gene regulation in vivo. Here we present evidence that the basic repeating units of eukaryotic chromatin, nucleosomes, are depleted from active regulatory elements throughout the Saccharomyces cerevisiae genome in vivo. We found that during rapid mitotic growth, the level of nucleosome occupancy is inversely proportional to the transcriptional initiation rate at the promoter. We also observed a partial loss of histone H3 and H4 tetramers from the coding regions of the most heavily transcribed genes. Alterations in the global transcriptional program caused by heat shock or a change in carbon source resulted in an increased nucleosome occupancy at repressed promoters, and a decreased nucleosome occupancy at promoters that became active. Nuclease-hypersensitive sites occur in species from yeast to humans and result from chromatin perturbation. Given the conservation of sequence and function among components of both chromatin and the transcriptional machinery, nucleosome depletion at promoters may be a fundamental feature of eukaryotic transcriptional regulation.

Classification of multiple cancer types by multicategory support vector machines using gene expression data

MOTIVATION High-density DNA microarray measures the activities of several thousand genes simultaneously and the gene expression profiles have been used for the cancer classification recently. This new approach promises to give better therapeutic measurements to cancer patients by diagnosing cancer types with improved accuracy. The Support Vector Machine (SVM) is one of the classification methods successfully applied to the cancer diagnosis problems. However, its optimal extension to more than two classes was not obvious, which might impose limitations in its application to multiple tumor types. We briefly introduce the Multicategory SVM, which is a recently proposed extension of the binary SVM, and apply it to multiclass cancer diagnosis problems. RESULTS Its applicability is demonstrated on the leukemia data (Golub et al., 1999) and the small round blue cell tumors of childhood data (Khan et al., 2001). Comparable classification accuracy shown in the applications and its flexibility render the MSVM a viable alternative to other classification methods. SUPPLEMENTARY INFORMATION http://www.stat.ohio-state.edu/~yklee/msvm.htm

A mixture model approach for the analysis of microarray gene expression data

Microarrays have emerged as powerful tools allowing investigators to assess the expression of thousands of genes in different tissues and organisms. Statistical treatment of the resulting data remains a substantial challenge. Investigators using microarray expression studies may wish to answer questions about the statistical significance of differences in expression of any of the genes under study, avoiding false positive and false negative results. We have developed a sequence of procedures involving finite mixture modeling and bootstrap inference to address these issues in studies involving many thousands of genes. We illustrate the use of these techniques with a dataset involving calorically restricted mice.

Transcriptional profiles associated with aging and middle age-onset caloric restriction in mouse hearts

To provide a global analysis of gene expression in the aging heart, we monitored the expression of 9,977 genes simultaneously in 5- and 30-month-old male B6C3F1 mice by using high-density oligonucleotide microarrays and several statistical techniques. Aging was associated with transcriptional alterations consistent with a metabolic shift from fatty acid to carbohydrate metabolism, increased expression of extracellular matrix genes, and reduced protein synthesis. Caloric restriction (CR) started at 14 months of age resulted in a 19% global inhibition of age-related changes in gene expression. Interestingly, CR also resulted in alterations in gene expression consistent with preserved fatty acid metabolism, reduced endogenous DNA damage, decreased innate immune activity, apoptosis modulation, and a marked cytoskeletal reorganization. These observations provide evidence that aging of the heart is associated with specific transcriptional alterations, and that CR initiated in middle age may retard heart aging by inducing a profound transcriptional reprogramming.

Gene expression profiling of aging using DNA microarrays.

We have previously employed high density oligonucleotide arrays representing thousands of genes to determine the gene expression profile of the aging process in skeletal muscle (gastrocnemius) and brain (cerebellum and neocortex) of male C57BL/6 mice. Specific gene expression profiles are associated with the aging process of individual organs, and caloric restriction can prevent or retard the establishment of these gene expression alterations. The use of DNA microarrays may provide a new tool to measure biological age on a tissue-specific basis and to evaluate at the molecular level the efficacy of interventions designed to retard the aging process.

The lifespan of Korean eunuchs

Summary Although many studies have shown that there are trade-offs between longevity and reproduction, whether such trade-offs exist in humans has been a matter of debate [1,2]. In many species, including humans, males live shorter than females, which could be due to the action of male sex hormones. Castration, which removes the source of male sex hormones, prolongs male lifespan in many animals, but this issue has been debated in humans [3]. To examine the effects of castration on longevity, we analyzed the lifespan of historical Korean eunuchs. Korean eunuchs preserved their lineage by adopting castrated boys. We studied the genealogy records of Korean eunuchs and determined the lifespan of 81 eunuchs. The average lifespan of eunuchs was 70.0 ± 1.76 years, which was 14.4–19.1 years longer than the lifespan of non-castrated men of similar socio-economic status. Our study supports the idea that male sex hormones decrease the lifespan of men.

Age and vitamin E-induced changes in gene expression profiles of T cells.

T cells are vulnerable to age-associated changes. Vitamin E has been shown to improve T cell functions in the old. We studied gene expression profiles of T cells to better understand the underlying mechanisms of age and vitamin E-induced changes in T cell function. Young and old C57BL mice were fed diets containing 30 (control) or 500 (supplemented) ppm of vitamin E for 4 wks. Gene expression profiles of T cells were assessed using microarray analysis with/without anti-CD3/anti-CD28 stimulation. Genes associated with cytokines/chemokines, transcriptional regulation, signal transduction, cell cycle, and apoptosis were significantly up-regulated upon stimulation. Higher SOCS3 and lower growth factor independent 1 (Gfi-1) expression in old T cells may contribute to age-associated decline in proliferation. Higher Gadd45 and lower Bcl2 expression may contribute to increased apoptosis in old T cells. Vitamin E supplementation resulted in higher expression of genes involved in cell cycle regulation (Ccnb2, Cdc2, Cdc6) in old T cells. Vitamin E supplementation resulted in higher up-regulation of IL-2 expression in young and old T cells and lower up-regulation of IL-4 expression in old T cells following stimulation. These findings suggest that aging has significant effects on the expression of genes associated with signal transduction, transcriptional regulation, and apoptosis pathways in T cells, and vitamin E has a significant impact on the expression of genes associated with cell cycle and Th1/Th2 balance in old T cells. Further studies are needed to determine whether these changes are due to the effects of aging at a single-cell level or to the shift in the ratio of naïve:memory T cells with age.

Variations in urinary 1-hydroxypyrene glucuronide in relation to smoking and the modification effects of GSTM1 and GSTT1

The measurement of the pyrene metabolite, 1-hydroxypyrene, in human urine has been used to assess recent exposure to polycyclic aromatic hydrocarbons (PAH). The objective of this study was to see whether genetic polymorphisms in metabolic enzymes could explain some of the variation in urinary 1-hydroxypyrene glucuronide (1-OHPG) excretion in relation to smoking. Forty-seven male hospital workers, who were not occupationally exposed to PAH, participated in this study. The urine samples were analyzed for 1-OHPG utilizing immunoaffinity chromatography and synchronous fluorescence spectroscopy. The analysis of GSTM1 and GSTT1 polymorphism was performed by PCR. The 1-OHPG concentration in the urine of the hospital workers was 0.57 +/- 0.85 micromol/mol creatinine, and ranged from 0.02 to 5.04 mciromol/mol creatinine. Cigarette smoking was significantly correlated with urinary 1-OHPG (r = 0.3976, P = 0.0056). The 1-OHPG excretion in GSTM1-deficient smokers was higher than that in GSTM1-positive smokers. On the other hand, 1-OHPG excretion was higher in GSTT1-positive smokers than in GSTT1-deficient smokers. It is important to note the variability of individual PAH metabolite excretion due to different GSTM1 and GSTT1 genotypes.

Comparison of three analytical methods for 1-hydroxypyrene glucuronide in urine after non-occupational exposure to polycyclic aromatic hydrocarbons

Urinary pyrene metabolites, 1-OHP and 1-OHPG, have been used as biomarkers for the assessment of occupational and environmental exposure to PAHs. This study compares the sensitivity and applicability of the different analytical methods of 1-OHPG for human biomonitoring of low level exposure to PAHs. Three analytical methods were compared: (1) HPLC method from that reported by Singh et al. (Singh, R., Tucek, M., Maxa, K., Tenglerova, J., Weyand, E.H., 1995. A rapid and simple method for the analysis of 1-hydroxypyrene glucuronide: a potential biomarker for polycyclic aromatic hydrocarbon exposure. Carcinogenesis 16, 2909-2915); (2) IAC-SFS method: the rapid and simple assay using IAC purification using monoclonal antibody specific for PAH-DNA adduct and PAH metabolites and SFS quantitation; and (3) IAC-HPLC method: IAC and HPLC separation and quantitation. The correlation between the IAC-SFS method, HPLC method, and the IAC-SFS method was determined in 20 first year-grade junior high school students (age 12-13) from Yochon, Korea who participated in a nationwide survey for the environmental disease surveillance projects in Korea. Chromatograms obtained by the IAC purification and HPLC quantitation method were clear with no interfering peaks adjacent to 1-OHPG, thus 1-OHPG could be easily quantitated. However, the HPLC method produced chromatogram profiles with many interfering peaks adjacent to 1-OHPG peak. The concentrations of 1-OHPG in 20 urine samples were similar when analyzed by all three analytical methods. The correlation coefficient between the IAC-HPLC and IAC-SFS methods was 0.915, and between the IAC-HPLC and HPLC methods was 0.844, and between the IAC-SFS and HPLC methods was 0.805. The analytical methods for 1-OHPG compared in this study showed a good correlation with one another. These results suggest that any of the methods can be applied to human biomonitoring of PAH exposure. However, SFS quantitation after IAC purification is rapid and simple because this method does not need HPLC separation of 1-OHPG.