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PubMed2005

Distribution of CIAPIN1 in normal fetal and adult human tissues.

CIAPIN1, a newly identified antiapoptotic molecule that plays an essential role in mouse definitive hematopoiesis, is considered a downstream effector of the receptor tyrosine kinase-Ras signaling pathway. Our previous studies have indicated that CIAPIN1 is involved in the development of multidrug resistance (MDR) in gastric cancer cells. However, the mechanism of CIAPIN1-mediated antiapoptosis and MDR has not been fully elucidated. To reveal the possible physiological role of CIAPIN1, we examined the expression and distribution of CIAPIN1 in fetal and adult human tissues using immunohistochemistry. We found that CIAPIN1 was ubiquitously distributed in fetal and adult tissues, and was localized in both the cytoplasm and the nucleus. The expression patterns of CIAPIN1 were similar in fetal and adult tissues, and was correlated with the previously described expression pattern of p21ras. These observations suggest that CIAPIN1 expression appears to be involved in cell differentiation, and that it might exert universal and possibly important physiological functions under the regulation of Ras in humans.

Adult↗
PubMed2005

Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.

Amino Acid Sequence↗
PubMed2006

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.

Antibodies, Monoclonal↗
PubMed2006

Breast tissue microarrays.

Tissue microarrays have been used effectively to study representative tissue from large groups of patients, with minimal technical and reagent costs. The construction of these arrays may appear complex, but with the use of a semiautomated tissue arrayer and a degree of manual dexterity, symmetrical, high-density arrays can be produced. Here, we highlight where problems in the construction, cutting, and evaluation of tissue microarrays can occur and how these can be prevented.

Breast↗
PubMed2007

Optimizing the integration of immunoreagents and fluorescent probes for multiplexed high content screening assays.

Immunoreagents formed the basis of early fixed end point high content screening (HCS) assays and their use in HCS applications in drug discovery will continue to increase. One important application of immunoreagents is their incorporation into multiplexed HCS assays in which multiple physiological features are simultaneously measured and related in the same cells. However, creating multiplexed HCS assays that incorporate multiple immunoreagents presents issues such as reagent compatibility, spectral signal overlap, and reproducibility that must be addressed. Here, an example multiplexed fixed end point HCS assay is used to guide potential assay developers on how to optimize complex, yet cellular information rich, multiplexed HCS assays although avoiding some common pitfalls.

Antibodies↗
PubMed2007

A pharmaceutical company user's perspective on the potential of high content screening in drug discovery.

It is early to fully reflect on the state of the art in high content screening (HCS), because it is still a relatively new approach in drug discovery. Although the development of the first microscopes are a century old and the first confocal microscope is only 20 yr old, the fluorescent probes used within HCS along with the combination of robotic automation and integrated software technologies are quite new. HCS will require a few more years to fully demonstrate its potential power in drug discovery. Within the last year, however, one has seen this ever-expanding field lure participants in from all areas of science, introducing newer versions of instruments and reagents such that the combined efforts result in platforms and tools that meet many organizational goals in multiple ways. The potential of HCS today lies in its versatility. HCS can be used for primary screening, basic research, target identification, biomarkers, cytotoxicity, and helping to predict clinical outcomes. HCS is being applied to stem cells, patient cells, primary hepatocytes, and immortalized cultured cells. We have noted for individual specialized assays, there are multiple solutions just as there are for those standardized universally accepted assays. Whether we have needed to query cellular processes under live conditions or wanted to follow kinetically the course of a compound's effects on particular cellular reactions, we have been hampered by only a few limitations. This chapter offers a glimpse inside the use of HCS in our drug discovery environment.

Animals↗
PubMed2007

Caged substrates applied to high content screening: an introduction with an eye to the future.

The use of photoremovable protecting groups in biology affords the end user high temporal, spatial, and concentration control of reagents and substrates. High content screening and other large-scale biology applications would benefit greatly from these advantages. Herein, we report progress in this field by highlighting the recent development of controllable siRNA (csiRNA), which is a dormant siRNA that can be activated using 365 nm light. Two different experimental designs are described to highlight the temporal and concentration variables that can be controlled. First, the RNAi process is activated at two timepoints, 24- and 48-h post-transfection, to demonstrate that the action of csiRNA does not begin until activated. Second, increasing light dosage exposure to cells transfected with csiRNA that controls the concentration of active siRNA molecules. All experiments are conducted in a 96-well format with light delivered through the UCOM device.

Actins↗
PubMed2007

Large-scale data management for high content screening.

High content screening (HCS) plays an important role in target selection in primary and secondary screening, but further developments in informatics and data management are needed for strategic implementation of HCS in the drug discovery process. An organization charter for the Research Informatics and Infrastructure Organization is described and is consists of four basic parts: Partner, Build Trust, Champion, and Core vs Noncore. The successful evolution of the charter over the last 5 yr is mapped using high-throughput screening and HCS data as an example. A future view of large-scale data management for the drug discovery process will incorporate all scientific information into multiple parameter type runs for many aspects of the science. This information will subsequently be aligned into a subset that an individual can digest and more easily choose the next appropriate steps.

Informatics↗
PubMed2007

Past, present, and future of high content screening and the field of cellomics.

High content screening (HCS) was created in 1996 to offer a new platform that could be used to permit relatively high-throughput screening of cells, in which each cell in an array would be analyzed at a sub-cellular resolution using multicolored, fluorescence-based reagents for both specificity and sensitivity. We developed HCS with the perspective of the history of the development of the automated DNA sequencers that revolutionized the field of genomics. Furthermore, HCS was based on a history of important developments in modern cytology. HCS integrates the instrumentation, application software, reagents, sample preparation, and informatics/bioinformatics required to rapidly flow from producing data, generating information, and ultimately creating new cellular knowledge. The HCS platform is beginning to have an important impact on early drug discovery, basic research in systems cell biology, and is expected to play a role in personalized medicine.

Animals↗
PubMed2007

Visualization of high content screening data.

Visualization is essential to the understanding of complex data derived from high content screening. It is necessary to present information in a way that captures patterns and trends in the data in order to answer specific questions while also providing a way to formulate new questions and hypothesis. Specific types of visualizations can provide information on the quality of the data, temporal, and spatial patterns of cellular response, cell phenotype, and the relationship to additional data such as the chemical structure of test compounds. Interacting with this data through linked visualizations and visual filtering facilitates exploration and hypothesis generation to better understand biological systems.

Image Processing, Computer-Assisted↗
PubMed2007

Linking microscopy and high content screening in large-scale biomedical research.

Microscopy has been a cornerstone of discovery in the academic life sciences for more than 100 yr. This comes from a unique ability to provide extremely rich information of biological structure and dynamics. The advent of digital imaging and machine vision has brought within itself the ability to collect images more easily and critically, the ability to measure objects and intensities within images. Although many continue to use microscopy in a qualitative manner, the analytical capabilities afforded by machine vision are increasingly being applied to basic cell biology and biomedical research. Scalable quantitative imaging technology might enable scientists and engineers to determine structure, dynamics, and function of entire biological systems rather than individual molecules or pathways. This chapter will provide an overview of early efforts in the academic community to apply high content screening to the study of biological systems.

Biomedical Research↗
PubMed2007

Systems biology in cancer research: genomics to cellomics.

Cancers result from large-scale deregulation of genes that lead to cancer pathophysiologies such as increase proliferation, decreased apoptosis, increased motility, increased angiogenesis, and others. Genes that influence proliferation and apoptosis are particularly attractive as therapeutic targets. To identify genes that influence these phenotypes, we have developed simple and rapid methods to measure apoptosis and cell proliferation using high content screening with YO-PRO-1 and anti-BrdU staining of BrdU pulsed cells, respectively.

Benzimidazoles↗
PubMed2007

High content screening as a screening tool in drug discovery.

In most pharmaceutical and biotechnology companies there is a need to always improve the quality of lead candidates. This demand resulted in the use of cell-based screening as a method of choice in drug discovery. High content screening (HCS) is multiplexed, functional cell-based screening. HCS can be used in all aspects of drug discovery as an engine for driving lead discovery. The biological applications of HCS have been implemented in research in signaling, cell shape changes and toxicology. HCS has enabled an insight in the cellular effects of our clinical candidates in multiple cellular phenomena like dual reporter assay, subcellular target translocation and cellular morphology. Discovery of therapeutic protein and small molecule converge on diseases in therapeutic areas such neurological disorders and autoimmune diseases. HCS is used for assay development, primary, secondary screening and toxicology testing. In this chapter, the use of HCS assays in drug discovery is described and highlight the necessary step to set-up successfully these assays for screening.

Animals↗
PubMed2007

Expression of TGF-beta signaling proteins in normal placenta and gestational trophoblastic disease.

The transforming growth factor beta (TGF-beta) is a vital regulator of placental development and functions. TGF-beta exerts several modulatory effects on trophoblast cells, such as inhibition of proliferation and invasiveness, and stimulation of differentiation by inducing multinucleated cell formation. In this study, we determine the expression patterns of TGF-beta signaling molecules in normal trophoblast, various hydatidiform mole types and choriocarcinoma. A total of 132 cases, including 51 normal placenta (20 first trimester, 11 second trimester, and 20 third trimester) and 81 gestational trophoblastic diseases (17 choriocarcinoma, and 64 hydatidiform moles: 39 complete, 6 partial, and 19 invasive) were immunohistochemically analyzed with anti-TGF beta1/2, TGF-beta receptor type I (TbetaRI), TbetaRII, Smad 2/3, and Smad 4 antibodies on paraffin blocks. In the case of normal placenta, maximal levels of all TGF-beta signaling molecules were observed in villous trophoblast in the first trimester, which decreased with gestational age. Expression of all the TGF-beta signaling proteins except Smad2/3, was significantly enhanced in various moles, relative to normal trophoblast. Moreover, TGF-beta signaling molecules were significantly downregulated in choriocarcinoma, compared to moles. In particular, TbetaRI and Smad2/3 levels were lower in choriocarcinoma than normal villous trophoblast (TbetaRI: p<0.025, Smad2/3: p<0.001). In conclusion, the TGF-beta signaling pathway plays an important role in the pathogenesis and progression of gestational trophoblastic disease, and may thus be employed as a potential therapeutic target and a diagnostic biomarker.

Activin Receptors, Type I↗
PubMed2005

Application of new tissue microarrayer-ZM-1 without recipient paraffin block.

The ZM-1 tissue microarrayer designed by our groups is manufactured in stainless steel and brass and contains many features that make TMA (tissue microarray) paraffin blocks construction faster and more convenient. By means of ZM-1 tissue microarrayer, biopsy needles are used to punch the donor tissue specimens respectively. All the needles with the punched specimen cylinders are arrayed into the array-board, with an array of small holes dug to fit the needles. All the specimen cylinders arraying and the TMA paraffin block shaping are finished in only one step so that the specimen cylinders and the paraffin of the TMA block can very easily be incorporated and the recipient paraffin blocks need not be made in advance, and the paraffin used is the same as that for conventional pathology purpose. ZM-1 tissue microarrayer is easy to be manufactured, does not need any precision location system, and so is much cheaper than the currently used instrument. Our method's relatively cheap and simple ZM-1 tissue microarrayer technique of constructing TMA paraffin block may facilitate popularization of the TMA technology.

Biopsy, Needle↗
PubMed2006

Selective spatial upregulation of intratumoral stromal aromatase in breast cancer patients: evidence for imbalance of local estrogen metabolism.

The suppression of local estrogens levels is of key importance in the treatment of ER-positive breast cancer. Essentially all endocrine strategies act by either suppressing estrogen formation or competitively inhibiting receptor-binding in tumor cells. Nevertheless, little is still known about the local expression of aromatase and sulfotransferase which are the key modulators of intra-tumoral estrogen levels. We have performed immunohistochemostry to investigate the expression of aromatase and sulfotransferase in 42 samples obtained directly from malignant breast tumors, and compared it to biopsies obtained from uninvolved tissue in the vicinity of the invasion front, and to distant breast tissue. We found that aromatase was equally detectable in both tumor epithelial and stroma, but was mostly epithelial in non-malignant tissues (P=0.00008, Fisher's exact test). Also, aromatase protein expression was significantly more common in tumoral stroma when compared with peritumoral and distant breast stroma (P=0.00005, and P<0.00001 respectively). With the notable exception of cystosarcoma phylloides, sulfotransferase protein was detectable only in epithelial tissues, regardless of the location within the diseased breast. However, epithelial sulfotransferase was correlated with epithelial aromatase (r=0.35461, P=0.0009, Spearman's rho test) and with the epithelial ER status (r=0.29313, P=0.005). We have demonstrated a differential aromatase and sulfotransferase protein expression pattern that is dependent on the spatial relation to a malignant breast tumor. Our results indicate a net increase in intratumoral active estrogen levels through increased stromal aromatization, while physiological local inactivation by sulfotransferase activity remains essentially unchanged.

Aromatase↗
PubMed2005

The usefulness of CDX-2 for differentiating primary and metastatic ovarian carcinoma: an immunohistochemical study using a tissue microarray.

Distinguishing primary ovarian carcinoma from metastatic carcinoma to the ovary is often difficult by histologic examination alone. Recently an immunohistochemical marker CDX-2 was found to be of considerable diagnostic value in establishing the gastrointestinal origin of metastatic tumors. The aim of this study was to determine whether CDX-2 can distinguish between these malignancies. Paraffin-embedded tissue sections from 57 primary ovarian tumors and 40 metastatic tumors to the ovary were immunostained for CDX-2, and results were compared to the ancillary immunohistochemical results for CK7/CK20, CEA, CA125, and her-2/neu. CDX-2 immunoreactivity was observed in most of metastatic carcinomas with colorectal (91%) and appendiceal (100%) origin, however CDX-2 was negative in all primary ovarian carcinomas, except for the mucinous subtype. Almost all primary ovarian carcinomas including the mucinous subtype showed diffuse and strong immunoexpression for CK7. CEA and CA125 were mainly found in metastatic and primary ovarian carcinoma, respectively. Her-2/neu overexpression was only noted in a small proportion of primary and metastatic ovarian carcinomas. These results suggest that CDX-2 is very useful immunohistochemical marker for distinguishing metastatic colorectal carcinoma to the ovary from primary ovarian carcinoma, including the mucinous subtype. Furthermore, combination with CDX-2 and CK7 strengthen the differential diagnosis between these tumors.

CA-125 Antigen↗