Differential Display of Gene Expression in Human Carcinomas

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5 Differential Display of Gene Expression in Human Carcinomas Roger S. Jackson II, Susanne Stein, Yong-Jig Cho, and Peng Liang

Introduction

of carcinogenesis are either applicable only to late-stage cancers or have lower predictive capability than desired, it is necessary to continue the search for new markers (Negm et al., 2002). Gene expression directs all aspects of cell biology, including apoptosis, cell cycle, development, differentiation, growth, homeostasis, and responses to the environment. Pathologic changes, such as those observed in human cancers, are driven most often by changes in gene expression resulting from epigenetic (methylation) and genetic mechanisms, such as acquisition of gene mutation(s), chromosomal alterations, and genomic instability (Hanahan et al., 2000). Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques are often employed to assess gene expression at the protein and mRNA levels, respectively, in combination with cellular localization (indicated by extracellular, cell surface, cytoplasmic, or nuclear staining patterns). Although effective, both techniques are laborious, time-consuming, and expensive processes used to screen for differential gene expression patterns and/or identify novel biomarkers of disease, such as cancer. With the exception of posttranscriptionally or post-translationally regulated genes, alterations in gene transcription generally correlate with alterations in gene translation (i.e., induction or

Cancer is a disease state caused by multiple genetic alterations that progressively lead to cellular transformation and unregulated cell proliferation (Hanahan and Weinberg, 2000). Disruption of normal cellular homeostasis through activating mutations of protooncogenes and inactivating mutations of tumor suppressor genes generally results in altered signal transduction. This can lead to sustained growth, disruption of genomic damage repair systems, evasion of programmed cell death (apoptosis), and an increased propensity at the stage of tumor development for angiogenesis, invasion, and metastasis (Gray et al., 2000; Hanahan et al., 2000). Although much has been learned to date about carcinogenesis, the molecular pathology of cancer remains to be elucidated fully. Oncologists and pathologists are continuously in search of novel cancer biomarkers—molecular signatures of a cellular phenotype that will aid in early cancer detection, pathological grading and staging, and risk assessment—in order to enable use and development of molecularly targeted therapies tailored to the patient, and to increase the rate of survival due either to earlier detection or improved therapies. Because most available biomarkers Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 1: Molecular Genetics; Lung and Breast Carcinomas

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Copyright © 2004 by Elsevier (USA) All rights reserved.

II Molecular Pathology

76 repression of an mRNA usually results in the corresponding induction or repression of the protein for which it encodes). Moreover, it is well known that altered signal transduction and/or gene expression often precedes morphological and histological changes that can be detected by IHC or ISH (Negm et al., 2002). The differential display method measures the expression of a gene transcript (steady-state mRNA levels), but does not assess protein expression, and thus may only imply a correlative change in the protein level based on gene induction or repression. Therefore, mRNA differential display can be employed as a quick and reliable primary screening strategy that will facilitate, focus, and complement further validation and characterization studies better done by techniques such as IHC or ISH. In this chapter, the procedure of differential display (DD) is presented, along with a discussion of some of the critical factors affecting the method’s accuracy.

Since its introduction in 1992, DD methodology traditionally has been based on 33P isotopic labeling of cDNA bands (Liang et al., 1992a). This is the most commonly used DD technology because of its sensitivity, simplicity, versatility, and reproducibility. Since its inception, numerous differentially expressed genes have been identified successfully across diverse biological systems and fields of study (reviewed by Liang and Pardee, 1995). More recently, the introduction of fluorescent primer labeling and robotic technology has enabled the establishment of an accurate, highthroughput fluorescent differential display (FDD) method (Figure 9) with similar sensitivity and reproducibility to the isotopic method, yet with greater accuracy (Cho et al., 2001). Either total or poly-A RNA can be used for DD. Although RNA isolated from cell lines is the easiest to obtain with uniformly high quality, methods have been

mRNA Population CAAAAAAAAAAA-An GAAAAAAAAAAA-An UAAAAAAAAAAA-An

I. Reverse Transcription

5′-AAGCTTTTTTTTTTTG-3′ (H-T11G) dNTPs MMLV reverse transcriptase CAAAAAAAAAAA-An GTTTTTTTTTTTCGAA

5′-AAGCTTGATTGCC-3′ (H-AP 1) 5′-R-AAGCTTTTTTTTTTTG-3′ (RH-T11G) dNTPs Taq DNA polymerase

II. PCR Amplification

AAGCTTGATTGCC

GTTTTTTTTTTTCGAA

AAGCTTGATTGCC

GTTTTTTTTTTTCGAA–R

AAGCTTGATTGCC

GTTTTTTTTTTTCGAA–R

III. Denaturing Polyacrylamide Gel & Fluorescent Image Scanning RNA Sample:

X

Y Negative electrode (–)

Positive electrode (+)

Figure 9 Schematic representation of fluorescent differential display. (Illustration courtesy of GenHunter Corporation, Nashville, TN.)

5 Differential Display of Gene Expression in Human Carcinomas devised to isolate RNA from fresh, frozen, and paraffinembedded tissues, such as tumors (referenced by Liang et al., 1995). Because it is essential that only high-quality RNA be used, all chromosomal DNA contamination must be removed from the RNA samples with DNase-I (with the addition of RNase inhibitor of RNase-free dH2O) before carrying out FDD. Other strategies have been developed to reduce the amount or RNA source material necessary to do a complete FDD screen, such as using a more proficient reverse transcriptase (Bosch et al., 2000), through preamplification of the cDNA pool from a limited number of cells (Jing et al., 2000; Zhao et al., 1998), or through the combination of a noncompetitive carrier molecule with the source RNA (Melichar et al., 2000). Ultimately, use of such techniques may result in the further reduction of the amount of mRNA required for a complete FDD screen to the lowest possible limit. This will likely enable increased use, in both clinical and molecular studies, of valuable clinical tissue specimens that are often limited by both accessibility and quantity. Moreover, the combination of material acquisition techniques (e.g., laser capture microdissection) with gene expression methods (e.g., FDD) may allow the long-awaited region-specific analysis of tissues and/or tumors. This could lend important insight into studies of local tumor interactions with the surrounding normal tissue and either remove or reduce the influence of tissue/tumor heterogeneity on studies conducted. The principle of FDD is to detect differential gene expression patterns by reverse transcription–polymerase chain reaction (RT-PCR). Using one of three one-base– anchored oligo-dT primers that anneal to the beginning of a subpopulation of the RNA poly-A tails, mRNA is reverse-transcribed into cDNA. This anchored oligo-dT (H-T11V) primer consists of 11 Ts (T11) with a 5′ HindIII (AAGCTT) site, plus one additional 3′ base V (where V may be dG, dA, or dC) that provides specificity. For FDD, fluorescent (rhodamine, red) labeled anchored R-H-T11V primers are combined with various arbitrary primers (H-AP primers; 13mer containing a 5′ HindIII site) in PCR steps. Amplified PCR products up to 700 bp can be separated on a denaturing polyacrylamide sequencing gel. The FDD image can be obtained using a fluorescent laser scanner. Side-by-side comparisons of cDNA patterns between or among relevant RNA samples would reveal differences in gene expression. The cDNA fragments of interest can be retrieved from the gel, purified, and reamplified with the same set of primers (lacking fluorescent labeling of H-T11V anchor primer) under the same PCR conditions as in the initial FDD-PCR reactions. For further molecular characterization, the obtained reamplified PCR fragments can be cloned and sequenced.

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DNA sequence analysis of these cDNA fragments by GenBank Blast search (www.ncbi.nlm.nih.gov/BLAST) may provide information as to whether a gene identified by FDD is a known, homologous to known, or novel gene. The final step of the FDD procedure is to confirm the differential expression of the obtained partial cDNAs by Northern Blot. The result of this analysis provides not only an independent confirmation of differential gene expression, but also information regarding the transcript size of the gene of interest. After confirmation, the cloned cDNA probe can be used to screen a cDNA library for a full-length clone, or as a starting point for rapid amplification of cDNA ends PCR (RACE-PCR), which is helpful for the functional characterization of the gene.

MATERIALS 1. RNApure Reagent (GenHunter, Nashville, TN). 2. MessageClean Kit (GenHunter), 10X reaction buffer: 10 mM Tris-Cl, pH 8.3, 50 mM potassium chloride (KCl), 1.5 mM magnesium chloride (MgCl2). 3. Diethyl pyrocarbonate (DEPC)-treated dH2O (GenHunter). 4. RNA loading dye (GenHunter). 5. 10X 3-(N-Morpholino)-propanesulfonic acid (MOPS) buffer: 0.4-M MOPS, pH 7.0, 0.1 M sodium acetate, 0.01 M disodium ethylenediaminetetraacetic acid (EDTA), store at room temperature in the dark. 6. 5X reverse transcription buffer: 125 mM TrisCl, pH 8.3, 188 mM KCl, 7.5 mM MgCl2, 25 mM dithiothreitol (DTT) (GenHunter). 7. Murine Moloney Leukemia Virus (MMLV) reverse transcriptase (100 units/μl) (GenHunter). 8. Deoxyribonucleotide triphosphate (dNTP) mix (2.5 mM) (GenHunter). 9. H-T11V anchor primer (V = A, C, G) (2 μM) (GenHunter). 10. R-H-T11V anchor primer (V = A, C, G) (8 μM) (GenHunter). (Note: Rhodamine-labeled primers are light sensitive.) 11. H-AP 13mer primers (1 to 160) with 50–70% GC content (2 μM) (GenHunter). 12. 10X PCR buffer: 100 mM Tris-HCl, pH 8.4, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin (GenHunter). 13. FDD loading dye: 99% formamide, 1 mM EDTA, pH 8.0, 0.009% xylene cyanol FF, 0.009% bromophenol blue (GenHunter). 14. Rhodamine locator dye (GenHunter). 15. 10X Tris-Boric acid-EDTA (TBE) buffer (for 1L): 108 g Trizma base, 55 g boric acid, 3.7 g EDTA. 16. Autoclaved millipure deionized H2O (dH2O). 17. Taq DNA polymerase (5 units/μl) (Qiagen).

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78 18. 20X Saline-sodium citrate (SSC) buffer: 3 M sodium chloride, 0.3 M sodium citrate, pH 7.0. 19. 20X Saline-sodium phosphate-EDTA (SSPE) (1L): 3.6 M sodium chloride, 0.2 M sodium dihydrophosphate, 0.02 M EDTA, pH 7.4. 20. 50X Denhardt’s solution (for 500 mL; store at −20°C): 5 g Ficoll 400, 5 g polyvinylpyrrolidone (molecular weight 360000), 5 g bovine serum albumin (BSA) fraction V. 21. Hybridization buffer: 5X SSPE, 50% formamide, 5X Denhardt’s solution, 0.1% sodium dodecyl sulfate (SDS) (store at −20°C), to which heat-denatured sheared nonhomologous salmon sperm DNA is added to 100 μg/ml just before use. 22. HotPrime DNA labeling Kit (GenHunter).

METHOD RNA Isolation from Cell Cultures Total RNA can be isolated with a one-step acidphenol extraction method using the RNApure Reagent. 1. For example, remove the cell culture medium from p150 cell culture plates (Sarstedt), wash with 10–20 ml cold 1X phosphate buffer saline (PBS), and set the plates on ice. Lyse the cells by adding 2 ml RNApure reagent to each plate, spread the solution across the plates, and incubate on ice for 10 min. Scrape the cells off the plate using a cell scraper, and pipette the cell lysate into sterile 1.5-ml Eppendorf tubes. Add 150 μl chloroform per milliliter of cell lysate, and mix well by vortexing for about 10 sec. Freeze the tubes at −80°C or proceed to step 2. 2. Spin the tubes at maximum speed (14,000g) in an Eppendorf centrifuge at 4°C for 10 min. 3. Carefully remove the upper (aqueous, RNAcontaining) phase and save into a new sterile Eppendorf tube. 4. Precipitate the RNA by adding an equal volume of 100% isopropanol to the aqueous phase, mix well by vortexing, and incubate on ice for 10 min. Spin the RNA down at maximum speed for 10 min at 4°C. Rinse the RNA pellet with 0.5–1 ml cold 70% ethanol (made with DEPC-dH2O). Spin down at maximum speed again for 10 min at 4°C. Remove the ethanol and resuspend the RNA pellet in 20–50 μl DEPC-dH2O. Make RNA aliquots and store at −80°C. 5. Before treatment with DNase-I, measure the RNA concentration at OD260 with a spectrophotometer, and check the integrity (18S and 28S rRNA bands) of the RNA samples by running 2 μg of each RNA sample on a 1% agarose 7% formaldehyde gel in 1X MOPS buffer.

DNase I Treatment of Total RNA Removal of all contaminating chromosomal DNA from the RNA sample is absolutely essential for successful DD. The MessageClean Kit is specifically designed for the complete digestion of single and double-stranded DNA. 1. Incubate 50 μl (10–50 μg) of total cellular RNA (use DEPC-dH2O when diluting RNA) with 10 units (1 μl) of DNase-I (RNase free) in 5.7 μl 10X reaction buffer for 30 min at 37°C. 2. Inactivate DNase-I by adding an equal volume of phenol:chloroform (3:1) to the sample. Mix by vortexing and leave the sample on ice for 10 min. Centrifuge the sample at maximum speed in an Eppendorf centrifuge for 5 min at 4°C. 3. Save the supernatant and ethanol precipitate the RNA by adding 3 volumes of ethanol and 0.1 volumes 3 M sodium acetate pH 5.2. 4. After incubation at −80°C for 1 hr (overnight to a few days at −80°C is recommended), pellet the RNA by centrifuging at maximum speed at 4°C for 10 min. Rinse the RNA pellet with 0.5 ml of 70% ethanol (made with DEPC-dH2O) and dissolve the RNA in 20 μl of DEPC-dH2O. 5. Measure the RNA concentration at OD260 with a spectrophotometer. Check the integrity of the RNA samples before and after cleaning with DNase-I by running 2-μg samples of each RNA on a 1% agarose 7% formaldehyde gel in 1X MOPS buffer. It is recommended to store the RNA samples as 1–2 μg aliquots at −80°C before using for DD to minimize freeze-thaw cycles and preserve RNA stability.

Reverse Transcription of mRNA The success of the differential display technique is dependent on the integrity of the RNA and that it is free of chromosomal DNA contamination. Upon completion of DNase-I treatment of the RNA, the mRNA then can be reverse transcribed into cDNA. 1. Set up three reverse transcription reactions for each RNA sample in three PCR tubes (0.2–0.5 ml size, thin-walled). Each should contain one of the three different one-base-anchored H-T11V primers (where V may be A, C, or G). For a final volume of 20 μl, combine: 9.4-μl dH2O, 4-μl 5X reverse transcription buffer, 1.6-μl dNTP mix (2.5 mM), 2-μl total RNA (0.1 μg/μl freshly diluted in dH2O), and 2-μl H-T11V primer (2 μM). To minimize pipetting errors, it is recommended to use a core mix without an RNA template for each anchored oligo-dT primer, especially if two or more RNA samples are to be compared.

5 Differential Display of Gene Expression in Human Carcinomas 2. Program the thermocycler to 65°C for 5 min, 37°C for 60 min, 75°C for 5 min, and 4°C for 5 min. 3. Add 1 μl MMLV reverse transcriptase to each tube 10 min after incubation begins at 37°C in order to initiate the RT reaction. At the end of the reaction, spin the tubes briefly to collect condensation. Set tubes on ice for FDD-PCR or store at −20°C for later use.

Fluorescent Differential Display The RNAspectra Red Kit can be used for this step as well as the previous reverse transcription reactions (also see Notes 1 to 3 at end of chapter). This step can be automated with a robotic liquid handling workstation such as the BioMek 2000 (Beckman), which can significantly increase the throughput and accuracy. 1. Set up on ice (in dim light) a 20 μl PCR reaction in thin-walled reaction tubes. For each primer set combination, use the following formula: 4.2 μl dH2O, 2 μl 10X PCR buffer, 1.6-μl dNTP mix (2.5 mM), 8 μl H-AP-primer (2 μM), 2 μl R-H-T11V (2 μM) (it has to contain the same H-T11V primer used for RT-PCR), 2 μl of a completed RT-PCR reaction mix (template), and 0.2 μl Taq DNA polymerase. Use core mixes as much as possible to avoid pipetting errors. 2. Mix well by pipetting up and down. PCR conditions are as follows: 94°C for 20 sec, 40°C for 2 min, 72°C for 1 min. After 40 cycles, follow with 72°C for 5 min and 4°C for 5 min. After completion of PCR, store the samples in the dark and either on ice or at −20°C until ready to run the gel. 3. Prepare a 6% denaturing polyacrylamide sequencing gel in 1X TBE buffer. Let the gel polymerize for about 2 hr before use. It is recommended that one glass plate be treated with Sigmacote (Sigma, St. Louis, MO) to facilitate the separation of the plates after running. Pre-run the gel for 30 min to prepare it for sample loading. The wells must be flushed completely just before sample loading as removal of urea from the wells after the pre-run is critical. 4. Mix each PCR reaction with 8 μl FDD loading dye and incubate at 80°C for 2–3 min immediately before loading onto the gel. 5. Electrophorese for 2 hr at 60 W constant power until the xylene dye (the slower-moving dye) reaches the bottom. 6. After the gel is run, clean the exterior of the plates well with water and 70% ethanol. 7. Scan the gel on a fluorescent laser scanner using a 585-nm filter (for Rhodamine). 8. Cut out the bands of interest after careful separation of the glass plates. For orientation of the lanes,

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it is very helpful to use the Rhodamine locator dye during scanning.

Purification and Reamplification of cDNA Bands from FDD 1. Soak the FDD gel slice (containing the cDNA band of interest) in 1 ml dH2O for 30 min, mixing gently by finger-tipping. 2. Remove the water without taking the gel slice, and add 50 μl dH2O. Boil the tube with its cap closed (and parafilm sealed) for 15 min to elute the DNA from the gel slice. Spin the tubes after cooling for 2 min at maximum speed to collect condensation and pellet the gel. Transfer the supernatant to a new tube and keep for the reamplification reaction. The tube with the gel slice can also be saved for the reamplification reaction. 3. Reamplification PCR should be carried out in a total volume of 40 μl using the same primer combination and concentration (4 μl of each 2-μM primer), but with an anchor primer (H-T11V) lacking the fluorescent label. The PCR conditions also should be kept the same, except for the dNTP concentration, which is changed, so use 1 μl, 250 μM dNTP mix instead. The following can be used as DNA templates: a) 4–5 μl of supernatant (step 2) and/or b) the gel slice (step 2), which still contains small traces of the removed DNA. 4. Check 30 μl of each PCR sample on a 1.5% agarose gel stained with ethidium bromide. Save the remaining PCR samples at −20°C for future experiments (e.g., cloning, Northern blot). Compare the size of the reamplified PCR products with that originally found on the FDD gel. Extract the positive reamplified cDNA fragments from the agarose gel using the Qiaex II Gel Extraction Kit (Qiagen).

Sequencing and Cloning of PCR Products One crucial advantage of FDD is the rapid identification of the cDNA sequence by direct sequencing of the PCR products without subcloning these fragments. Alternatively, after gel purification, reamplified cDNA probes can be ligated into various cloning vector systems and then subjected to DNA sequence analysis.

Confirmation of Differential Gene Expression by Northern Blot Confirmation of differential gene expression patterns identified by FDD can be obtained through the

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80 use of independent methods such as Northern blot. Northern blots are extremely sensitive and provide additional details such as the transcript size of the gene of interest through the use of the cDNA fragments isolated from the FDD screen as probes. Northern blots can be performed following the standard procedure (Asubel et al., 1993) with the use of the HotPrime DNA labeling Kit. Although DD can be used to detect amplification, reduction, or loss of gene expression, one should be reminded that this method is unlikely to detect mutations at the DNA level directly. For diseases caused by single gene mutations that have a clear genetic component, chromosome mapping of the mutation locus should be a method of choice. It should be emphasized that the method is only a simple screening tool. Upon confirmation by Northern blot, however, the differentially expressed cDNA probe(s) might release a series of molecular studies leading to a better understanding of complex pathways.

Applications of Fluorescent Differential Display Although FDD has the power and ease to assess differential gene expression between multiple related cell lines or tissues, the achievement of physiologically relevant results is highly dependent on the quality of the experimental system implemented with all necessary controls. For example, gene expression studies by DD or FDD can be done using RNA isolated from normal and aberrant tissues of patients with the same disease; from different developmental stages (such as tumor progression from normal epithelium to benign adenoma, carcinoma in situ, and metastatic carcinoma); along a temporal course of treatment or between different related treatments in a cell line of interest; and so on (Liang et al., 1995). The FDD technique is most successful when the RNA samples to be compared are from closely matched, homologous populations of primary cells or cell lines (Martin et al., 1999). Proper experimental design will allow quicker identification, isolation, and functional studies of genes that may be important for the process of interest, while simultaneously eliminating false positives that may randomly vary between experimental groups assessed (Liang et al., 1995). Sideby-side comparison of RNA from many different yet related treatment groups may shed light on specific processes or pathways involved, rather than just simply being informative about differential gene expression.

RESULTS AND DISCUSSION Differential display is a globally used method for identifying differentially expressed genes in eukaryotic cells. Ease of use and rapid results makes fluorescent differential display an ideal screening tool to use in order to focus and complement clinical and diagnostic studies that traditionally use IHC and/or ISH techniques. Unlike IHC and ISH, which are sporadic screening methods based on the availability of antibodies or riboprobes, DD is a systematic screening of all expressed genes in a cell through the use of multiple arbitrary and anchor primer combinations. The DD technique also allows identification of new genes and potential biomarkers for which no antibodies or probes are available (i.e., novel and rare messages can be identified). Indeed, DD has proven useful particularly for finding diagnostic markers for pathological processes in which altered gene expression has been shown to play a role. Examples of such biomarkers identified by DD include cyclin G, Mob-1, and macrophage lectin (Liang et al., 1997). The DD procedure sets the stage for IHC and/or ISH studies because it allows detection of the known, homologous to known, or novel genes of interest for the process or system being studied that then can be followed up by IHC or ISH for further characterization. The DD method has been used successfully by many groups in the study of human carcinomas, including those of the breast, colon, and prostate (reviewed by Gray et al., 2000; Guan et al., 2000; Martin et al., 1998). More specifically, the DD method has been used to identify tumor promotor (oncogenes) or tumor suppressor genes, candidate biomarkers, and cell cycle regulated genes (reviewed by Gray et al., 2000; Martin et al., 1998). In addition, our lab and others have used differential display to identify and clone genes regulated by Ras (e.g., Wang et al., 2002; Zhang et al., 1997; Zhang et al., 1998) and p53 (reviewed in Stein et al., 2002; Stein et al., 2003), both of which are mutated frequently in carcinomas. Our comprehensive FDD screening using hundreds of FDD primer combinations yielded more than two dozen inducible or repressible p53-regulated genes (to be published elsewhere), in addition to the detection of known target genes, including p21waf1 (Figure 10), HDM2, and PIG3. This provides strong validation of our nonbiased and exhaustive screening strategy for p53 target genes by FDD. Furthermore, FDD also allows the analysis of digital gene expression profiling and precise quantification of gene expression differences, unlike IHC or ISH (Cho et al., 2001; Stein et al., 2003). Interestingly, more than 50% of these genes

5 Differential Display of Gene Expression in Human Carcinomas

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A2 G20

p53-3

G54

Ap21-1

G116

8+ 12+ 8– 12– 8+ 12+ 8– 12– 8+ 12+ 8– 12–

Figure 10 Differential gene expression of p53 and p21waf1 in A2 and p53-3 cell lines. A: Examples of fluorescent displays from comprehensive screening of A2 (tet-off p53 DLD-1 colon carcinoma) and p53-3 (tet-off p53 H1299 lung adenocarcinoma) cell lines showing induction of p53 and p21 after removal of tetracycline (tet) at different time points (8 hr and 12 hr). Displays of p53 used the HT11G anchor primer with H-AP primers 20 and 54 for A2 and 116 for p53-3 cells (since the vector-imposed 3′tail of p53 in the constructs used for these two cell lines are different from each other and from that of endogenous wild type p53). Arrows are used to indicate the inducible p53 band or p21 bands (doublet) for each display. As a positive control, p21 induction was displayed with a p21-specific 13mer primer (p21-1) in combination with HT11A anchor primer. B: Northern blots results of p21 induction in A2 and p53-3 cells done through the use of a human p21-specific cDNA probe (474 bp) with equivalent loading (10-μg total RNA per lane) demonstrated by 28S and 18S rRNA band intensities. C: Western analysis of p53 and p21 protein levels in A2 and p53-3 cells. Antibodies used include the antip21 antibody (C-19, Santa Cruz Biotechnology, CA) and a polyclonal pAB 1801 anti-p53 antibody. As a control for equivalent protein loading (50-μg total protein per lane), antiactin antibody was used (A2066, Sigma-Aldrich, St. Louis, MO).

Ap21-1

8+ 12+ 8– 12–

hours after

8+ 12+ 8– 12– tetracycline removal

A A2 + tet

A2 – tet

p53-3 + tet

p53-3 – tet

8

8

8

8

12

12

12

hours after tetracycline removal

12

p21

2.1 kb 28S

rRNA 18S

B

C

A2 + tet

A2 – tet

p53-3 + tet

p53-3 – tet

8

8

8

8

12

12

16

16

hours after tetracycline removal

53 kD

p53

21 kD

p21

42 kD

Actin

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82 represent novel and/or previously uncharacterized genes (to be published elsewhere). This is in contrast to methodology of DNA microarrays, which can recognize only gene sequences present on the chip and may be limited because of improper cross-hybridization. Other advantages of DD over DNA microarrays are the requirement of much less RNA, the ability to compare more than two different RNA samples simultaneously, and also the ability to detect rare mRNAs. In summary, our results and those from other colleagues provide evidence that DD is an elegant methodology to identify and quantify changes in gene expression, elucidate candidate biomarkers, and discover novel genes involved in important biological and pathological pathways.

Notes 1. All materials or products for DD and FDD technology are commercially available from GenHunter Corporation (Nashville, TN). The company also offers automated FDD services (from reverse transcription reactions to FDD results). 2. The DD method is widely used to identify and isolate differentially expressed genes, and now the automated FDD method can increase the throughput of screening for many more differentially expressed genes. At the moment, some problems may appear because of flaws in the technical equipment. For example, check the glass plates for evenness as this is a critical requirement for a correct scan. Also, the comb must fit well between the two glass plates to avoid lane leakage. The major drawback to FDD is the expense of a fluorescent laser scanner. 3. Information to minimize extrinsic and intrinsic factors can be found in some recently published papers or reviews (Cho et al., 2002; Liang et al., 1995; Liang, 1998).

Acknowledgments This work was supported in part by NIH grants CA76969 and CA74067 to P.L., by a grant from the Deutsche Akademie der Naturforscher-Leopoldina (Halle, Germany) to S.S., and by a NIH Cellular, Biochemical, and Molecular Sciences Training Grant #GM08554-7 to R.J. The authors greatly appreciate the help of E. Thomas in the proofreading of the manuscript. We thank GenHunter Corporation (Nashville, TN) for permission to adapt the protocol from its RNASpectra Fluorescent Differential Display kit and for use of the Hitachi FMBIO II fluorescent laser scanner. We also thank Dr. C. Prives for the tet-off regulated p53 H1299 (p53-3) cell line, Dr. J. Pietenpol for the p53 pAB1801 antibody, and Dr. B. Vogelstein for the tet-off regulated p53 DLD-1 (A2) cell line.

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