Journal of Histochemistry and Cytochemistry, Vol. 49, 925-926, July 2001, Copyright © 2001, The Histochemical Society, Inc.


BRIEF REPORT

Monitoring Signal Transduction in Cancer: From Chips to FISH

Robert A. Lerscha, Jingly Fungb, H.-Ben Hsieha, Jan Smidac, and Heinz-Ulrich G. Weiera
a Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, California
b School of Medicine, University of California, San Francisco, California
c Arbeitsgruppe Zytogenetik, gsf-Forschungszentrum für Umwelt und Gesundheit, Neuherberg, Germany

Correspondence to: Robert A. Lersch, Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 74-157, Berkeley, CA 94720. E-mail: ralersch@lbl.gov


  Summary
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The microarray format of RNA transcript analysis should provide new clues to carcinogenic processes. Because of the complex and heterogeneous nature of most tumor samples, histochemical techniques, particularly RNA fluorescent in situ hybridization (FISH), are required to test the predictions from microarray expression experiments. Here we describe our approach to verify new microarray data by examining RNA expression levels of five to seven different transcripts in a very few cells via FISH. (J Histochem Cytochem 49:925–926, 2001)

Key Words: spectral imaging, FISH, microarray, RNA, cancer


  Introduction
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OUR LABORATORY is adapting microarray technology to identify new genes involved in the formation of tumors in the thyroid, breast, and prostate. Previously, the transcription levels of only a few genes could be assayed per experiment. Microarrays circumvent this limitation. However, a second problem limits progress in cancer research. Most tumors are a mix of cell types, due in part to the normal complexity of the tissue and in part to the complexity of tumors as they evolve from benign to metastatic. If researchers collect microarray data without confronting the problem of tumor heterogeneity, important correlations will be missed.

Preliminary studies performed in our lab and elsewhere indicated that solid tumors are heterogeneous with respect to oncogene expression. Fig 1 shows the expression of brk (O'Bryan et al. 1991 ) in a childhood thyroid cancer that arose after the Chernobyl nuclear accident (Zitzelsberger et al. 1999 ). In this case, two cells strongly express brk (arrows point to their nuclei) and a nearby cell does not express brk. These studies were performed with touchprep papillary thyroid tumor specimens using filter-based microscope systems and only two different cDNA probes. If more than two probes are used per experiment, more slides, time, and reagents must also be used. All experiments involving human cells or cell lines were approved by the U.C. Berkeley Human Subject IRB.



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Figure 1. Frozen tissue touchprep of childhood thyroid tumor (S246) was hybridized with a cDNA probe for brk. The probe was labeled with digoxigenin and detected with rhodamine-conjugated antibodies. About 5% of the cells expressed brk at high levels, the other cells at low levels. Arrows indicate two high brk-expressing cells.

To address this problem, we are developing a system to simultaneously measure cell-by-cell RNA levels of several different markers. The proposed scheme to discriminate benign and malignant neoplasms and to identify new prognostic markers will take advantage of new methods and our experience with oncogene activation in thyroid tumors. Our short-term goal is to develop a system which determines the relative level of expression of five tyrosine kinase genes using FISH-based methods and Spectral Imaging (SIm). Existing SIm instrumentation can record fluorescence spectra from 400 nm to 1100 nm with about 10-nm resolution, whereas the resolution of a light microscope is about 1 µm.

SIm combines the techniques of fluorescence microscopy, charge-coupled device (CCD) camera, and Fourier spectroscopy. The light emitted from each point of the sample is collected with the microscope objective and sent to a collimating lens. This light travels through an optical head (interferometer), is focused on a CCD camera, and the resulting data are processed with a computer. The interferometer divides each incoming beam (light from the microscope) into two coherent beams and creates a variable optical path difference (OPD) between them. The beams are then combined to interfere with each other. The resulting interference intensity is measured by the CCD detector as a function of the OPD. The intensity vs OPD is an "interferogram." The spectrum, i.e., intensity, as a function of wavelength can be recovered from the interferogram using Fourier transforms. The first published application of SIm (spectral karyotyping or SKY) (Schroeck et al. 1996 ) was developed to screen metaphase spreads for translocations with 24 chromosome-specific whole chromosome painting probes labeled with Spectrum Green, Spectrum Orange, Texas Red, Cy5, or Cy5.5, or combinations thereof. The multiple bandpass filter set (Chroma Technology; Brattleboro, VT) used for fluorochrome excitation was designed to provide broad emission bands (giving a fractional spectral reading from ~450 nm to ~850 nm).

Although we can use much of the existing technology, the ratio-labeling color scheme for SKY will not work for RNA detection because different labeled cDNA probes might co-localize. Therefore, we will label each cDNA probe with a unique reporter. The fluorescence spectra of the different reporter molecules can partially overlap, because the signals can be resolved by a computer algorithm termed "Spectral Un-Mixing (SUN)" (Applied Spectral Imaging; Carlsbad, CA). SUN enables us to deconvolute overlapping spectra and recover single component images from the spectral image.

The existing commercial SKY metaphase chromosome analysis software will be modified to increase its automated signal processing, RNA identification in interphase cells, integration of cDNA probe signals, and databasing of results. We will only refine the analysis software; the acquisition methods of capturing a spectral image and a separate high-contrast monochrome DAPI image will remain unchanged. A prototype of this system for analysis of protein markers has already been developed (Tsurui et al. 2000 ) and will aid our efforts to analyze RNA transcript levels.

Initially we will hybridize cDNA probes to brk, ret, c-met, trk, axl/ufo (obtained from Research Genetics; Huntsville, AL) using five cyanine dyes (Amersham; Arlington Heights, IL). On the basis of previous microarray results, we will also add members of the Eph tyrosine kinase family. cDNA probes will be prepared by incorporating fluorochrome-labeled deoxynucleoside triphosphates by random priming or PCR amplification (Weier et al. 1994 , Weier et al. 1995 ). We will also build a reference spectra database for these probes. Standard fixation and hybridization protocols should detect the target RNAs. It is unclear if we will need to prepare custom blocking agents to DNA repeats in the 3'-untranslated region of some of our targets. Using artificial mixtures of existing cell lines, we will develop software modules needed to measure intracellular levels of five RNA species and to determine important process parameters: sensitivity, accuracy, and reproducibility.


  Footnotes

Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 2–7, 2001.


  Acknowledgments

Supported by a grant from the Director, Office of Science, Office of Biological and Environmental Research, US Department of Energy, under Contract DE-AC03-76SF00098, by the Cancer Research Foundation of America and grants from the Cancer Research Program, US Army Medical Research and Material Command, US Department of the Army (DAMD17-99-1-9250, PC991359).

Received for publication December 4, 2000; accepted February 16, 2001.


  Literature Cited
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Summary
Introduction
Literature Cited

O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, Espinosa R, III, Le Beau MM, Earp HS, Liu ET (1991) axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 11:5016-5031[Medline]

Schroeck E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson–Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T (1996) Multicolor spectral karyotyping of human chromosomes. Science 273:494-497[Abstract]

Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T (2000) Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition. J Histochem Cytochem 48:653-662[Abstract/Free Full Text]

Weier H-U, Polikoff D, Fawcett JJ, Greulich KM, Lee K-H, Cram S, Chapman VM, Gray JW (1994) Generation of five high complexity painting probe libraries from flow sorted mouse chromosomes. Genomics 24:641-644

Weier H-UG, Wang M, Mullikin JC, Zhu Y, Cheng J-F, Greulich KM, Bensimon A, Gray JW (1995) Quantitative DNA fiber mapping. Hum Mol Genet 4:1903-1910[Abstract]

Zitzelsberger H, Lehmann L, Hieber L, Weier H-UG, Janish C, Fung F, Negele T, Spelsberg F, Lengfelder E, Demidchik E, Salassidis K, Kellerer AM, Werner M, Bauchinger M (1999) Cytogenetic changes in radiation-induced tumors of the thyroid. Cancer Res 58:135-140[Abstract]





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