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Genome-Wide Expression Profiling of 8-Chloroadenosine- and 8-Chloro-Camp-Treated Human Neuroblastoma Cells Using Radioactive Human Cdna Microarray

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Genome-Wide Expression Profiling of 8-Chloroadenosine- and 8-Chloro-Camp-Treated Human Neuroblastoma Cells Using Radioactive Human Cdna Microarray EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 34, No. 3, 184-193, July 2002 Genome-wide expression profiling of 8-chloroadenosine- and 8-chloro-cAMP-treated human neuroblastoma cells using radioactive human cDNA microarray Gil Hong Park1, Jaegol Choe2, involved in proliferation and transformation (trans- Hyo-Jung Choo1, Yun Gyu Park1, forming growth factor-β, DYRK2, urokinase-type Jeongwon Sohn1, and Meyoung-kon Kim1,3 plasminogen activator and proteins involved in tran- scription and translation) which were in close paral- lel with those by 8-Cl-cAMP. Our results indicated 1Department of Biochemistry, College of Medicine, Korea University, that the two drugs shared common genomic path- Seoul, 136-701, Korea ways for the down-regulation of certain genes, but 2Department of Nuclear Medicine, College of Medicine, Korea Uni- used distinct pathways for the up-regulation of dif- versity, Seoul, 136-701, Korea ferent gene clusters. Based on the findings, we sug- 3Corresponding author: Tel, +82-2-920-6184; gest that the anti-cancer activity of 8-Cl-cAMP Fax, +82-2-923-0480; E-mail, [email protected] results at least in part through 8-Cl-adenosine. Thus, the systematic use of DNA arrays can provide Received 30 May , 2002 insight into the dynamic cellular pathways involved in anticancer activities of chemotherapeutics. Abbreviations: 8-Cl-adenosine, 8-chloro-adenosine; 8-Cl-cAMP, 8- chloro-cyclic adenosine 3,5-monophosphate; PKA, protein kinase Keywords: 8-Cl-adenosine, 8-Cl-cAMP, anticancer activ- A; RFXAP, regulatory factor X-associated protein; DCC, deleted in ity, radioactive cDNA microarray, genome-wide expres- colorectal cancer; CDKs, cyclin-dependent kinases; MHC, major sion histocompatibility complex; TGF-β, transforming growth factor-β; DYRK2, dual specificity tyrosine phosphorylated and regulated kinase 2 Introduction 8-Cl-adenosine, dephosphorylated metabolite of anti- Abstract neoplastic agent 8-Cl-cAMP has a growth-inhibitory effect, which is produced from 8-Cl-cAMP by the Previous reports raised question as to whether 8- action of serum phosphodiesterase and 5-nucleotidase chloro-cyclic adenosine 3,5-monophosphate (8-Cl- activity (Figure. 1) (Taylor et al., 1990; Lange-Carter et cAMP) is a prodrug for its metabolite, 8-Cl-adenos- al., 1993; Halgren et al., 1998). 8-Cl-cAMP, a site- ine which exerts growth inhibition in a broad spec- selective cAMP analogue, exhibits growth inhibition in a trum of cancer cells. The present study was carried broad spectrum of cancer cell lines. However, its out to clarify overall cellular affects of 8-Cl-cAMP mechanism of action remains unelucidated. One and 8-Cl-adenosine on SK-N-DZ human neuroblas- possible mode of action is to induce cytotoxicity by acting toma cells by systematically characterizing gene as competitive inhibitor in biochemical pathway involving expression using radioactive human cDNA microarray. cAMP-dependent protein kinase [protein kinase A (PKA)] Microarray was prepared with PCR-amplified cDNA (Cho-Chung, 1989; Cho-Chung et al., 1995; Noguchi et of 2,304 known genes spotted on nylon membranes, al., 1998). PKA is a serine/threonine protein kinase that employing 33P-labeled cDNAs of SK-N-DZ cells as a is activated by cAMP. Two isozymes of PKA, PKA-I and probe. The expression levels of approximately 100 PKA-II, are distinguished by the association of type R cDNAs, representing about 8% of the total DNA ele- subunit (R) or type R subunit (R) with common C ments on the array, were altered in 8-Cl-adenosine- or subunits, respectively. Four different R subunits-RIa, 8-Cl-cAMP-treated cells, respectively. The genome- RIb, RIIa, and RIIb-have been identified (Taylor et al., wide expression of the two samples exhibited partial 1990). Expression of the PKA RIa subunit is correlated overlaps; different sets of up-regulated genes but with active cell growth and transformation, and increased the same set of down-regulated genes. 8-Cl-adenos- in various human tumors and cell lines. Furthermore, ine treatment up-regulated genes involved in differ- overexpression of the PKA RIa subunit is correlated with entiation and development (LIM protein, connexin malignancy and poor prognosis in cancer patients 26, neogenin, neurofilament triplet L protein and (Miller et al., 1993a; Miller et al., 1993b; Simpson et al., p21WAF1/CIP1) and immune response such as natural 1996; Miller et al., 1997). In contrast, a high level of Rb killer cells protein 4, and down-regulated ones is correlated with growth arrest and differentiation (Cho- cDNA microarray characterizes 8-Cl-adenosine-treated neuroblastoma 185 Figure 1. Metabolic conversion of 8-Cl-cAMP to 8-Cl-adenosine Chung, 1990). Cancer cells treated with 8-Cl-cAMP Materials and Methods showed a decrease of Ra and an increase of Rb, leading to inhibition of cancer cell growth (Ally et al., Cell culture and RNA preparation 1988; Ciardiello et al., 1990; Rohlff et al., 1993). SK-N-DZ cells (ATCC #CRL-2149) grown in RPMI 1640 The mechanism of 8-Cl-cAMP action is not yet medium with 10% fetal bovine serum, 50 units/ml penicillin, clarified as to whether it is a prodrug for its metabolite, 500 µg/ml streptomycin, and 1 mM glutamine (GIBCO) 8-Cl-adenosine. On the one side, 8-Cl-cAMP exerts its were seeded at 5x105 cells per dish at humidified growth-inhibitory effect by binding to PKA in a competitive o atmosphere of 5% CO2 at 37 C. Cells were counted on manner against cAMP (Rohlff et al., 1993; Noguchi et a Coulter Counter, and were treated with 5 µM 8-Cl- al., 1998) and on the other side, the effect of 8-Cl-cAMP adenosine (Biology Life Science Institute, Bremen, is at least in part due to its metabolite 8-Cl-adenosine. Germany) or 8-Cl-cAMP (National Cancer Institute, Drug Furthermore, the anticancer effect of 8-Cl-cAMP has Synthesis and Chemistry Branch, Bethesda, MD. USA) been suggested to be independent of residual activity of for 24 h, respectively. Total RNA was prepared by using PKA or concentration of cAMP (Lange-Carter et al., RNeasy Midi Kit (Qiagen, Chatsworth, CA, USA). 1993; Cummings et al., 1996; Langefeld et al., 1997; Halgren et al., 1998; Carlson et al., 2001; Yin et al., Human cDNA microarray 2001). A human cDNA microarray was primarily derived from a In order to evaluate the molecular portrait of anti- cancer actions of 8-Cl-adenosine versus 8-Cl-cAMP, we commercially available master set of approximately have selected human cDNA microarray comprised of 15,000 human verified-sequences (Research Genetics, 2,304 nonredundant human clones to examine whole Inc.). The 15,000 human cDNA clone set was sorted for spectrum of genes affected by both drugs. The a list of genes (2,304 elements) representing families such microarray-based genomic survey is one of the high- as differentiation, development, proliferation, transformation, throughput well known approaches, and has allowed cell cycle progression, immune response, transcription parallel studies on expression patterns of thousands of and translation factors, oncogenes, and molecules genes (Schena et al., 1995; Duggan et al., 1999; Konu involved in cell growth and maintenance. PCR-amplified et al., 2001; Vawter et al., 2001). In the present study, cDNAs were spotted on nylon membranes. The general we investigated genome-wide expression profiling under methodology of arraying is based on the procedures of the treatment of 8-Cl-adenosine or 8-Cl-cAMP as DeRisi et al. (1996). compared to untreated control. As a cancer cell model, SK-N-DZ human neuroblastoma cell line was chosen, cDNA Radiolabeling because it has higher concentration of PKA than other Total RNAs prepared from SK-N-DZ cells treated with 8- types of cancer cells (Cho et al., 2000). Cl-adenosine or 8-Cl-cAMP and untreated control cells 186 Exp. Mol. Med. Vol. 34(3), 184-193, 2002 were used to synthesize 33P-labeled cDNAs by reverse control cells was calculated by comparing the Z score transcription. Briefly, 3~10 µg RNA were labeled in a differences (Z differences) among the same genes. This reverse transcription reaction containing 5X first strand facilitates to compare each gene that had been up- or PCR buffer, 1 µg of 24-mer poly dT primer, 4 µl of 20 downregulated as compared to the control cells. Z mM each dNTP excluding dCTP, 4 µl of 0.1 M DTT, 40 differences were calculated first by subtracting Z scores U of RNase inhibitor, 6 µl of 3000 Ci/mmol α-33P dCTP of the control from each Z score of the samples. These to a final volume of 40 µl. The mixture was heated at differences were normalized again to distribute their 65oC for 5 min, followed by incubation at 42oC for 3 min. position by subtracting the average Z difference and Two µl (specific activity: 200,000 U/ml) of Superscript dividing with the standard deviation of the Z differences. reverse transcriptase (Life Technologies) was then These distributions represent the Z ratio value, and added and the samples were incubated for 30 min at provide the efficiency for comparing each microarray 42oC, followed by the addition of 2 µl of Superscript experiment (Vawter et al., 2001). reverse transcriptase and another 30 min of incubation. Spotfire produced scatter plots of intensity values. Five µl of 0.5 M EDTA was added to chelate divalent (Spotfire, Inc., Cambridge, MA) (Tanaka et al., 2000). cations. After the addition of 10 µl of 0.1 M NaOH, the Cluster analysis was performed on Z-transformed samples were incubated at 65oC for 30 min to hydrolyze microarray data by using two programs available as remaining RNA. Following the addition of 25 µl of 1 M shareware from Michael Eisens laboratory (http:// Tris (pH 8.0), the samples were purified using Bio-Rad rana.lbl.gov).
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