Aldo Massimi

Making and reading microarrays

There are a variety of options for making microarrays and obtaining microarray data. Here, we describe the building and use of two microarray facilities in academic settings. In addition to specifying technical detail, we comment on the advantages and disadvantages of components and approaches, and provide a protocol for hybridization. The fact that we are now making and using microarrays to answer biological questions demonstrates that the technology can be implemented in a university environment.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-β

Epithelial–mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-β (TGF-β) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-β-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-β within 4 hours after treatment. TGF-β-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell–matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-β. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

Multiple sclerosis: Re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-β1 (TGF-β1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-β1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.

MicroRNA‐23b cluster microRNAs regulate transforming growth factor‐beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads

Transforming growth factor‐beta / bone morphogenetic protein (TGFβ/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFβ ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR‐23b cluster miRNAs comprising miR‐23b, miR‐27b, and miR‐24‐1 and miR‐10a, miR‐26a, and miR‐30a as up‐regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR‐23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR‐23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFβ signaling molecules, as putative miR‐23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3′ UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down‐regulation of constructs containing Smad 3, 4, or 5, 3′ UTRs by a mixture of miR‐23b cluster mimics. Knockdown of miR‐23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC‐3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR‐23b mimics during bile duct differentiation of HBC‐3 cells blocked the process. Conclusion: Our data provide a model in which miR‐23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down‐regulating Smads and consequently TGFβ signaling. Concomitantly, low levels of the miR‐23b miRNAs are needed in cholangiocytes to allow TGFβ signaling and bile duct formation. (HEPATOLOGY 2009.)

Injecting new ideas into microarray printing.

Microarray technology is changing the way biologists think about genetic networks and interactions between genes. Although the reductionist approach is crucial as a starting point in understanding the potential function of a given gene, it is clear that the ultimate result of a gene product is not a summation of a simple linear pathway, but the net interaction of multiple gene products and pathways. This holistic approach requires high-throughput analysis tools. To be truly useful, the system used to produce microarrays needs to address issues such as sensitivity, reproducibility, specificity, density, and cost. In this issue, Yamomoto and colleagues1 describe a method for the generation of microarrays using the Bubble Jet variation of ink-jet technology developed by Canon (Tokyo). Their noncontact method may be more reliable than current contact printing approaches and also has the potential to substantially increase the density of probes deposited on the microarray surface. Currently, two formats are in wide use for microarrays: the Affymetrix (Santa Clara, CA) system, based on a photolithographic oligonucleotide synthesis technology, and the robotic printing of cDNA on a chemically treated microscope slide. Affymetrix’s approach has the advantage of being an integrated package, providing chip sets, protocols, automated processing stations, and data analysis software. The chips provide an extensive set of controls allowing chip-tochip comparisons. The ease of use of the package and its sensitivity were the two factors most liked by users of the Affymetrix system in a survey2 conducted by the Association of Biomolecular Resource Facilities (ABRF; Santa Fe, NM). One complaint, however, was the expense of Affymetrix chips, with an average cost per experiment of over

Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

2,000. A potential solution may come from a recent paper by Cerrina and colleagues3 that describes the use of computergenerated virtual masks for in situ synthesis of oligonucleotide-based microarrays without the need for premanufactured glass photolithographic masks. Since a major cost in the development of custom oligo arrays is the production of the masks (four times the number of bases in the oligonucleotides), this new method could lead to the development of more affordable custom oligonucleotide chips. Today, most microarrays used in academic research consist of cDNAs spotted onto treated microscope slides. These can be subdivided into those using pens to contact the slide surface and those using some other noncontact technique. The cDNA spotting system is more à la carte than the Affymetrix chip, with a wide selection of scanners, arraying robots, and analysis software. Whereas outsourcing a microarray experiment through a company could cost

Classification of DNA methylation patterns in tumor cell genomes using a CpG island microarray.

4,000 or more per experiment, an in-house facility can produce chips at a tiny fraction of that cost. The reproducibility of cDNA arrays depends upon several factors, such as the viscosity of the printing solutions, the precision of the robotics, the quality of the printing pens, humidity, dust, and slide quality. Contact printing tends to produce somewhat irregular spots, and there is a practical limit to spot size that can be obtained. Any change in the distance between the printing pens and the print surface will also cause chip-to-chip variations. The results presented by Yamamoto and coworkers in this issue suggest that a noncontact print method with an active ejection method like an ink jet is far less susceptible to these effects. The goals of microarray research are to have a chip with all genes of an organism on it, and to be able to get reliable signals using as little RNA as possible. Current protocols require reasonably large amounts of total RNA on chips with 10,000 spots in a 4 cm2 area. New labeling protocols will produce probes that can yield similar signal intensities using only a tenth to a twentieth of the amount of starting RNA. To cover the 100,000 or more genes in a human or mouse would require either several 4 cm2 chips or the entire surface area of a slide, resulting in a substantial increase in the volume of hybridization solution needed. If the size of the spots on a chip were reduced Thomas M. Harris is research associate, Aldo Massimi is associate, and Geoffrey Childs is professor at the department of molecular genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (childs@aecom.nyu.edu). Figure 1. Spot the difference. (A) A pen tip assembly for cDNA spotting. (B) Non-contact printing using the Bubble Jet type print head

Genetic/genomic analysis of signal transduction pathways using cDNA microarray technology

Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT24-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods.

MAT a set of microarray data management and analysis tools

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample’s response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.

Abstract 5706: High-throughout library screening identifies phytochemical inducers of phase II metabolism enzymes GSTP1 and NQO1 in human lung cells

cDNA microarray technology provides a novel approach to identify individual target genes and survey global genetic programs under the control of specific signalling pathways in mammalian systems. Smad2 and Smad3 are highly homologous members of the receptor-regulated subfamily of Smad proteins with a central role in TGF- signalling and target gene regulation.