Detection of Caenorhabditis elegans Germ Cell Apoptosis Following Exposure to Environmental Contaminant Mixtures: A Crude Oil-Dispersant Mixture Example.

Crude oil disasters, such as the Deepwater Horizon accident, have caused severe environmental contamination and damage, affecting the health of marine and terrestrial organisms. Some previous studies have demonstrated cleanup efforts using chemical dispersant induced more potent toxicities than oil alone due to an increase in bioavailability of crude oil components, such as PAHs. However, there still lacks a systematic procedure that provides methods to determine genotypic and phenotypic changes following exposure to environmental toxicants or toxicant mixture, such as dispersed crude oil. Here, we describe methods for identifying a mechanism of dispersed crude oil-induced reproductive toxicity in the model organisms, Caenorhabditis elegans (C. elegans). Due to the genetic malleability of C. elegans, two mutant strains outlined in this chapter were used to identify a pathway responsible for inducing apoptosis: MD701 bcIs39 [lim-7p::ced-1::GFP + lin-15(+)], a mutant strain that allows visualization of apoptotic bodies via a green fluorescent protein fused to CED-1; and TJ1 (cep-1(gk138) I.), a p53/CEP-1 defective strain that is unable to activate apoptosis via the p53/CEP-1 pathway. In addition, qRT-PCR was utilized to demonstrate the aberrant expression of apoptosis (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) and cytochrome P450 (cyp14a3, cyp35a1, cyp35a2, cyp35a5, and cyp35c1) protein-coding genes following exposure to dispersed crude oil. The procedure outlined here can be applicable to determine whether environmental contaminants, most of time contaminant mixture, cause reproductive toxicity by activation of the proapoptotic, p53/CEP-1 pathway.

Drug-dependent behaviors and nicotinic acetylcholine receptor expressions in Caenorhabditis elegans following chronic nicotine exposure.

Nicotine, the major psychoactive compound in tobacco, targets nicotinic acetylcholine receptors (nAChRs) and results in drug dependence. The nematode Caenorhabditis elegans' (C. elegans) genome encodes conserved and extensive nicotinic receptor subunits, representing a useful system to investigate nicotine-induced nAChR expressions in the context of drug dependence. However, the in vivo expression pattern of nAChR genes under chronic nicotine exposure has not been fully investigated. To define the role of nAChR genes involved in nicotine-induced locomotion changes and the development of tolerance to these effects, we characterized the locomotion behavior combining the use of two systems: the Worm Tracker hardware and the WormLab software. Our results indicate that the combined system is an advantageous alternative to define drug-dependent locomotion behavior in C. elegans. Chronic (24-h dosing) nicotine exposure at 6.17 and 61.7µM induced nicotine-dependent behaviors, including drug stimulation, tolerance/adaption, and withdrawal responses. Specifically, the movement speed of naïve worms on nicotine-containing environments was significantly higher than on nicotine-free environments, suggesting locomotion stimulation by nicotine. In contrast, the 24-h 6.17µM nicotine-treated worms exhibited significantly higher speeds on nicotine-free plates than on nicotine-containing plates. Furthermore significantly increased locomotion behavior during nicotine cessation was observed in worms treated with a higher nicotine concentration of 61.7µM. The relatively low locomotion speed of nicotine-treated worms on nicotine-containing environments also indicates adaption/tolerance of worms to nicotine following chronic nicotine exposure. In addition, this study provides useful information regarding the comprehensive in vivo expression profile of the 28 "core" nAChRs following different dosages of chronic nicotine treatments. Eleven genes (lev-1, acr-6, acr-7, acr-11, lev-8, acr-14, acr-16, acr-20, acr-21, ric-3, and unc-29) were significantly up-regulated following 61.7µM nicotine treatment, in which worms showed significantly increased locomotion behavior. This study provides insights into the linkage between nicotine-induced locomotion behavior and the regulation of nicotinic acetylcholine receptors.

Impacts of chronic low-level nicotine exposure on Caenorhabditis elegans reproduction: identification of novel gene targets.

Effects and mechanisms of chronic exposure to low levels of nicotine is an area fundamentally important however less investigated. We employed the model organism Caenorhabditis elegans to investigate potential impacts of chronic (24h) and low nicotine exposure (6.17-194.5 µM) on stimulus-response, reproduction, and gene expressions. Nicotine significantly affects the organism's response to touch stimulus (p=0.031), which follows a dose-dependent pattern. Chronic nicotine exposure promotes early egg-laying events and slightly increased egg productions during the first 72 h of adulthood. The expressions of 10 (egl-10, egl-44, hlh-14, ric-3, unc-103, unc-50, unc-68, sod-1, oxi-1, and old-1) out of 18 selected genes were affected significantly. Other tested genes were cat-4, egl-19, egl-47, egl-5, lin-39, unc-43, pink-1, and age-1. Changes in gene expression were more evident at low dosages than at relatively high levels. Genes implicated in reproduction, cholinergic signaling, and stress response were regulated by nicotine, suggesting widespread physiological impacts of nicotine.

Label-free classification of cultured cells through diffraction imaging.

Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells.

5-Fluorouracil drug alters the microRNA expression profiles in MCF-7 breast cancer cells.

5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for breast cancer treatment. Although aberrant expression of protein-coding genes was observed after 5-FU treatment, the regulatory mechanism remains poorly understood. MicroRNAs (miRNAs) are a newly identified class of small regulatory RNAs which play an important role in gene regulation at the post-transcriptional levels. Recent evidence suggests an important role of miRNAs in initiation, progression, and metastasis of human cancers. In this study, using a combined advanced microarray and quantitative real-time PCR (qRT-PCR) technology, we show that 5-FU significantly alters the global expression profile of miRNAs in vitro. After 48 h of treatment with a low dose (0.01 µM), 42 miRNAs were differentially expressed in MCF-7 breast cancer cell line. Of these, 23 miRNAs were up-regulated with up to 4.59-fold changes, while 19 were down-regulated with up to 1.89-fold changes. A majority of these miRNAs are associated with cancer development, progression, and metastasis. Target prediction and GO analysis suggest that these differentially expressed miRNAs potentially target many oncogenes, tumor suppressor genes and genes related to programmed cell death, activation of immune response, and cellular catabolic processes.

Evaluation and identification of reliable reference genes for pharmacogenomics, toxicogenomics, and small RNA expression analysis.

Pharmacogenomics, toxicogenomics, and small RNA expression analysis are three of the most active research topics in the biological, biomedical, pharmaceutical, and toxicological fields. All of these studies are based on gene expression analysis, which requires reference genes to reduce the variations derived from different amounts of starting materials and different efficiencies of RNA extraction and cDNA synthesis. Thus, accurate normalization to one or several constitutively expressed reference genes is a prerequisite to valid gene expression studies. Although selection of reliable reference genes has been conducted in previous studies in several animals and plants, no research has been focused on pharmacological targets, and very few studies have had a toxicological context. More interestingly, no studies have been performed to identify reference genes for small RNA analysis although small RNA, particularly microRNA (miRNA)-related research is currently one of the fastest-moving topics. In this study, using MCF-7 breast cancer cells as a model, we employed quantitative real-time PCR (qRT-PCR), one of the most reliable methods for gene expression analysis in many research fields, to evaluate and to determine the most reliable reference genes for pharmacogenomics and toxicogenomics studies as well as for small RNA expression analysis. We tested the transcriptional expression of five protein-coding genes as well as five non-coding genes in MCF-7 cells treated with five different pharmaceuticals or toxicants [paclitaxel (PTX), gossypol (GOS), methyl jasmonate (JAS), L-nicotine (NIC), and melamine (mela)] and analyzed the stability of the selected reference genes by four different methods: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method. According to our analysis, a protein-coding gene, hTBCA and four non-coding genes, hRNU44, hRNU48, hU6, and hRNU47, appear to be the most reliable reference genes for the five chemical treatments. Similar results were also obtained in dose-response and time-course assays with gossypol (GOS) treatment. Our results demonstrated that traditionally used reference genes, such as 18s RNA, ß-actin, and GAPDH, are not reliable reference genes for pharmacogenomics and toxicogenomics studies. In contrast, hTBCA and small RNAs are more stable during drug treatment, and they are better reference genes for pharmacogenomics and toxicogenomics studies. To widely use these genes as reference genes, these results should be corroborated by studies with other human cell lines and additional drugs classes and hormonal treatments.

MicroRNA expression profile of MCF-7 human breast cancer cells and the effect of green tea polyphenon-60.

This study reports for the first time the microRNA expression profile of human breast cancer MCF-7 cells and the effect of green tea. Although hundreds of miRNAs have been identified in humans, only a small proportion (25.6%) of miRNAs are expressed in MCF-7 cells. Low concentration treatment with Polyphenon-60 significantly alters the miRNA expression profile in MCF-7 cells. Twenty three miRNAs have been identified with differential expression after a 48 h treatment with 10 µg/ml Polyphenon-60 (green tea extract). These miRNAs include miR-21 and miR-27 that were found to be down-regulated following treatment with green tea. These two miRNAs have previously been identified as being overexpressed in MCF-7 breast cancer cells, with miR-21 specifically implicated in down-regulating the tumor suppressor gene, tropomyosin-1. This data supports the hypothesis that Polyphenon-60-induced modification of the breast cancer miRNA expression profile contributes to the efficacy of green tea treatment. The resulting decrease in carcinogenesis is further supported by the altered miRNA regulation of potential oncogenes and tumor-suppressor genes.

MicroRNA as a new player in the cell cycle.

MicroRNAs (miRNAs) are a newly identified class of small regulatory non-coding endogenous RNAs that are ubiquitous from animals to plants and have pivotal functions in nearly all biological and metabolic processes. Increasing evidence shows that miRNAs are also new players regulating many protein-coding genes and specific pathways during the cell cycle. This review focuses on the functions of miRNAs in the cell cycle of differentiated cells and embryonic stem (ES) cells. Aberrant expression of these cell-cycle-related miRNAs may result in carcinogenesis, revealing the potential of miRNAs as therapeutic targets for clinical purposes.

Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of BCL2 overexpressing immortalized breast cell line.

Bcl-2 is an anti-apoptotic protein that is frequently overexpressed in cancer cells but its role in carcinogenesis is not clear. We are interested in how Bcl-2 expression affects non-cancerous breast cells and its role in the cell cycle. We prepared an MCF10A breast epithelial cell line that stably overexpressed Bcl-2. We analyzed the cells by flow cytometry after synchronization, and used cDNA microarrays with quantitative reverse-transcription PCR (qRT-PCR) to determine differences in gene expression. The microarray data was subjected to two pathway analysis tools, parametric analysis of gene set enrichment (PAGE) and ingenuity pathway analysis (IPA), and western analysis was carried out to determine the correlation between mRNA and protein levels. The MCF10A/Bcl-2 cells exhibited a slow-growth phenotype compared to control MCF10A/Neo cells that we attributed to a slowing of the G(1)-S cell cycle transition. A total of 363 genes were differentially expressed by at least two-fold, 307 upregulated and 56 downregulated. PAGE identified 22 significantly changed gene sets. The highest ranked network of genes identified by IPA contained 24 genes. Genes that were chosen for further analysis were confirmed by qRT-PCR, however, the western analysis did not always confirm differential expression of the proteins. Downregulation of the phosphatase CDC25A could solely be responsible for the slow growth phenotype in MCF10A/Bcl-2 cells. Increased levels of GTPase Cdc42 could be adding to this effect. PAGE and IPA are valuable tools for microarray analysis, but protein expression results do not always follow mRNA expression results.

Nuclear respiratory factor 2 activates transcription of human mitochondrial translation initiation factor 2 gene.

We studied the transcriptional regulation of the human mitochondrial translation initiation factor 2 (IF2mt) gene. The minimal promoter region for the human IF2mt gene contains binding sites for Nuclear Respiratory Factor 2 (NRF-2), which is often involved in the transcription of mitochondrial-related genes. Electrophoresis mobility shift assay (EMSA) analyses indicated that NRF-2alpha/beta binds to the IF2mt promoter. Reporter assays, where HEK293T cells were co-transfected with an NRF-2alpha/beta-expressing vector and/or an IF2mt promoter reporter vector, revealed that NRF-2 trans-activates the IF2mt promoter. NRF-2 sites were also found in the promoters of several other mitochondrial translation factors, which suggests NRF-2 may play a key role in the regulation of mitochondrial protein synthesis.

The human mitochondrial translation initiation factor 2 gene (MTIF2): transcriptional analysis and identification of a pseudogene.

Mitochondrial translation initiation factor 2 (MTIF2) is nuclear-encoded and functions in mitochondria to initiate the translation of proteins encoded by the mitochondrial genome. To gain insight into mechanisms that regulate MTIF2 gene expression, the genomic copy and the 5' and 3' flanking regions of MTIF2 were isolated using a combination of genomic library screening and polymerase chain reaction (PCR). MTIF2 is approximately 33.5-kb long and contains 16 exons, confirming data from the Human Genome Project. With RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we mapped the transcription start point in human heart tissue to a cytosine residue 296 bp upstream from the translation initiation site. The region surrounding the transcription start point contains consensus binding sites for transcription factors Sp1, nuclear respiratory factor 2 (NRF-2) and estrogen receptor, while enhancer binding sites were identified upstream. Promoter constructs were prepared in a luciferase reporter vector and transiently transfected into 293T cells. The minimal promoter gave an expression level 3.5x higher than the SV40 control (P=0.001), while the construct containing the minimal promoter plus the enhancer region gave a 3.8x higher level of expression compared to the control (P<0.001). We also discovered a pseudogene of MTIF2 and mapped it to chromosome 1p13-12.