Darpp-32: a Novel Antiapoptotic Gene in Upper Gastrointestinal … · truncated isoform t-DARPP (AY070271) from adenocarcinoma of the stomach with 17q amplicon (6, 7). In addition,

Darpp-32: a Novel Antiapoptotic Gene in Upper

Gastrointestinal Carcinomas

Abbes Belkhiri,1Alexander Zaika,

1Nataliya Pidkovka,

1Sakari Knuutila,

3

Christopher Moskaluk,2and Wa’el El-Rifai

1

1Digestive Health Center of Excellence and 2Department of Pathology, University of Virginia Health System, Charlottesville, Virginia and3Departments of Pathology and Medical Genetics, Haartman Institute, University of Helsinki and Helsinki University Central HospitalLaboratory Diagnostics, Helsinki, Finland

Abstract

We show the molecular mechanisms involved in Darpp-32overexpression and its biological role in upper gastrointesti-nal adenocarcinomas (UGC). A tumor tissue array of 377samples was developed and used to detect DARPP-32 DNAamplification and protein overexpression, which occurred in32% and 60% of UGCs, respectively. Concomitant overexpres-sion of mRNA for Darpp-32 and its truncated isoform t-Darppwas observed in 68% of tumors (P < 0.001). When Darpp-32and t-Darpp were overexpressed in AGS and RKO gastroin-testinal cells, up to a 4-fold reduction in the apoptosis rate wasobserved (terminal deoxynucleotidyl transferase–mediatednick-end labeling and Annexin V assays) in response tocamptothecin, sodium butyrate, and ceramide. However, theintroduction of mutations in phosphorylation sites abrogatedthis effect. Expression of Darpp-32 and t-Darpp preserved themitochondrial transmembrane potential and was associatedwith increased levels of Bcl2 protein. A reversal of Bcl2 proteinlevel was obtained using small interfering RNAs for Darpp-32and t-Darpp. Luciferase assays using the p53 and p21 reporterplasmids and probing of immunoblots with antibodies specificfor p53 transcriptional targets, such as Hdm2 and p21,indicated that neither Darpp-32 nor t-Darpp interfere withp53 function. Altogether, we show more frequent mRNA andprotein overexpression of Darpp-32 than DNA amplification,suggesting that, in addition to amplification, transcriptionalor posttranscriptional mechanisms may play an importantrole. The expression of Darpp-32 and t-Darpp is associatedwith a potent antiapoptotic advantage for cancer cellsthrough a p53-independent mechanism that involves preser-vation of mitochondrial potential and increased Bcl2 levels.(Cancer Res 2005; 65(15): 6583-92)

Introduction

Upper gastrointestinal adenocarcinomas (UGC; i.e., adenocarci-nomas of the stomach and esophagus) are poorly responsive totherapy and have an unfavorable outcome (1). It has beenestimated that in the year 2000 alone, >1.3 million new cases ofUGCs were identified and >984,000 people died from them. Thiscombination of these cancers is therefore the most common formof incident cancer and the second most common cause of death

from cancer in the world (2, 3). UGCs are characterized by complexgenetic changes and frequent genomic amplification at 17q and20q; however, the critically important genes involved in thedevelopment of these tumors remain largely undefined (4).Darpp-32 is a neuronal protein that plays a central role in the

dopamine signaling pathway in the brain (specifically featured inthe citation for the Nobel Prize in Physiology or Medicine in 2000;ref. 5). We have recently cloned DARPP-32 (AF464196) and its noveltruncated isoform t-DARPP (AY070271) from adenocarcinoma ofthe stomach with 17q amplicon (6, 7). In addition, we and othershave recently documented the overexpression of Darpp-32 in avariety of common adenocarcinomas, such as those of the colon,esophagus, breast, and prostate (8, 9).Apoptosis (programmed cell death) is regulated by multiple

signaling pathways and plays a critical role in cancer initiationand development. It is well established that some oncogenicmutations can disrupt apoptosis, leading to neoplasia and tumorprogression (10). The BCL2 oncogene, which was first identifiedat the chromosomal breakpoint of chromosomal translocationst(8;14) (q24;q32) in a human leukemia cell line, promotes cellsurvival by blocking apoptosis (11). Genetic changes that affectapoptosis, such as overexpression of Bcl2 or loss of p53 function,can promote cancer development and are frequently found incancer cells. The expression of Bcl2 regulates the release ofproapoptotic mitochondrial proteins, including cytochrome c ,and eventually, activation of the caspases. This mechanism isrelated to many of the effects of this protein involved andunderscores a fundamental role for blocking apoptosis duringtumor progression (12–14).Drug resistance is a major problem that affects cancer therapy

and can be induced by several mechanisms. In addition to druginactivation (15, 16) and altered drug transport systems (17, 18),evasion of apoptosis plays a significant role (19). Overexpressionand oncogenic mutations of prosurvival signaling molecules, suchas epidermal growth factor receptor (20, 21), Akt (also known asprotein kinase B; ref. 22), nuclear factor-nB (23, 24), andantiapoptotic Bcl2 family members (25, 26), correlate with reducedresponse to chemotherapy.The molecular mechanisms underlying Darpp-32 expression, as

well as its biological role in cancer development and tumorprogression, are yet to be elucidated. In this study, we investigatedthe molecular mechanisms underlying Darpp-32 overexpression inUGCs. Because of the frequent tumor chemoresistance and poorprognostic outcome for patients with UGCs, we explored thebiological effect of Darpp-32 overexpression on apoptosis. Wefound that Darpp-32 and t-Darpp confer antiapoptotic effectagainst several drugs in a p53-independent fashion that involves anincrease of Bcl2 protein level and preservation of mitochondrialpotential.

Note: The contents of this work are solely the responsibility of the authors and donot necessarily represent the official views of the National Cancer Institute, Universityof Virginia, or University of Helsinki.

Requests for reprints: Wa’el El-Rifai, Digestive Health Center of Excellence,University of Virginia Health System, P.O. Box 800708, Charlottesville, VA 22908-0708.Phone: 434-243-6158; Fax: 243-6169; E-mail: [email protected].

I2005 American Association for Cancer Research.

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Materials and Methods

Tissue samples. A total of 301 UGCs and 76 normal stomach paraffin-embedded tissue samples were available for the fluorescent in situhybridization (FISH) and immunohistochemical analysis. In addition, 74frozen tumor samples and 15 normal gastric epithelial samples were dissectedfor optimal tumor content (>70%) and used for RNA extraction, cDNAsynthesis, and subsequent quantitative real-time reverse transcription-PCR(RT-PCR) assays. All tissue samples were collected in accordance withInstitutional Review Board–approved protocols. Tissues were stained withH&E and representative regions were selected for inclusion in a tissue array.Tissue cores with a diameter of 0.6 mm were retrieved from the selectedregions of the donor blocks and punched to the recipient block using a manualtissue array instrument (Beecher Instruments, Silver Spring, MD); sampleswere punched in duplicate. Control samples from normal epithelial specimenswere punched in each sample row. Sections (5 Am) were transferred topolylysine-coated slides (SuperFrostPlus, Menzel-Glaser, Braunschweig, Ger-many) and incubated at 37jC for 2 hours. The resulting tumor tissue array wasused for FISH and immunohistochemical analysis. All tumors and normalgastric mucosal epithelial tissues were histologically verified. The adenocarci-nomas collected ranged from well-differentiated (WD) to poorly differentiated(PD), stages I to IV, with a mix of intestinal and diffuse-type tumors.

Fluorescent in situ hybridization. Before hybridization, tumor tissuearray sections were deparaffinized and pretreated in a microwave oven (10minutes at 92jC in 0.01 mol/L citric acid). After rinsing in PBS, tissuesmounted on slides were digested with pepsin solution (Digest-All 3, ZymedLaboratories, Inc., South San Francisco, CA) for 10 minutes at 37jC, rinsedin PBS, dehydrated in graded ethanol series, and air-dried. To investigateDARPP-32 copy number amplification, we obtained the BAC clone CTD-2019C10 (Research Genetics, Huntsville, AL) and sequenced it to verify thatit contained the DARPP-32 gene. FISH analysis was employed to probe ourtissue arrays. The DARPP-32 probe was labeled with biotin-14-ATP (LifeTechnologies, Invitrogen Corp., San Diego, CA) by nick translation,precipitated with herring sperm DNA (0.62 Ag/AL, Sigma, St. Louis, MO)and human Cot-1 DNA (0.62 Ag/AL, Life Technologies), and dissolved inhybridization buffer (50% formamide, 20% dextran sulfate, 2� SSC). Thetissue arrays were denatured in 70% formamide/2� SSC (pH 7) at 75jC for5 minutes and the probes were denatured at 75jC for 5 minutes. The probeswere then applied onto the tissue array slides. For cytogenetic localizationof DARPP-32 , the probe was hybridized to a slide preparation with normallymphocyte culture metaphase spreads denatured in 70% formamide/2�SSC at 68jC for 2 minutes. The hybridization was done at 37jC for 2 days.Posthybridization washes were done at 45jC using a series of solutions(pH 7): 50% formamide, 2� SSC, twice in 0.1� SSC, and finally in 4� SSC/0.2% Tween. Signal was detected with fluorescein avidin and fluoresceinanti-avidin D (Vector Laboratories, Inc., Burlingame, CA). The slides weremounted with an antifading medium that contained 4V,6-diamidino-2-phenylindole (DAPI) counterstain (Vector Laboratories). The tumor arraypreparations were surveyed with a Zeiss Axiophat fluorescence microscope( for the fluorescence-labeled arrays) and an Olympus BH-2 lightmicroscope. A minimum of 50 nonoverlapping nuclei were scored fromeach case. FISH results were scored based on the number of signals per cellas follows: FISH score of 0 to 1 = 2 to 5 signals; a score of 2 = 6 to 9 signals; ascore of 3 = 10 to 15 signals; and a score of 4 = >15 signals. Amplificationwas defined as z6 signals (score z2) in z50% of cancer cells, or when largegene copy number clusters where detected.

Quantitative real-time reverse transcription-PCR. For quantitative

real-time RT-PCR, mRNA was isolated from 15 normal gastric mucosasamples and 74 primary UGCs, which included 22 distal adenocarcinomas

(antrum and body), 52 proximal adenocarcinomas (GEJ and lower

esophageal), using the RNeasy kit (Qiagen, Gmbh, Hilden, Germany).

Single-stranded cDNA was synthesized using the Advantage RT-for-PCR Kit(Clontech, Palo Alto, CA). Quantitative PCR was then done using an iCycler

(Biol-Rad, Hercules, CA), with a threshold cycle number as determined

using iCycler software version 3.0. Reactions were done in triplicate andthreshold cycle numbers were averaged. Gene-specific primers for DARPP-

32 and t-DARPP were designed and then obtained from GeneLink

(Hawthorne, NY; sequences available upon request). Results were normal-

ized to HPRT1 , which had minimal variation in all normal and neoplasticsamples tested and is considered a reliable and stable reference gene for

real-time RT-PCR (27). The fold overexpression was then calculated

according to the formula 2(Rt-Et)/2(Rn-En), as described previously (7).

Immunohistochemistry for Darpp-32. An avidin-biotin immunoper-

oxidase assay was done after pretreatment in a microwave with citrate

buffer for 20 minutes, and rabbit anti-Darpp-32 antibody (H-62; 1:200

dilution, Santa Cruz Biotechnology, Inc., California, CA) was applied at

room temperature. The antibody had been raised against a recombinant

protein corresponding to amino acids 134 to 195 at the COOH terminus of

Darpp-32. The immunostaining for the gastric mucosa samples from

healthy individuals lay between 0 and 1+. The immunoreactivity of the

samples tested was scored as 0, absent; 1+, weak (1-33%); 2+, moderate

(34-66%); 3+, strong (67-100%). Cases with 2+ or 3+ scores typically had

moderate-to-strong cytoplasmic immunoreactivity. None of the normal

samples had a FISH or immunohistochemistry score higher than 1.

Cell culture, vectors, transfection, and generation of stable celllines. Colonic (RKO, ATCC No. CRL-2577) and gastric (AGS, ATCC No. CRL-1739) cancer cells were maintained in DMEM and F-12 (HAM) media

(Invitrogen Life Technologies, Carlsbad, CA) , respectively, with 10% fetal

bovine serum (FBS), at 37jC in an atmosphere containing 5% CO2. The

expression plasmid for FLAG-Darpp-32 was generated by PCR amplificationof the full-length coding sequence of Darpp-32 cDNA and cloned in frame

into a modified pcDNA3.1Zeo (Invitrogen Life Technologies) plasmid

containing an oligonucleotide corresponding to the FLAG peptide(DYKDDDDK) inserted into the HindIII and EcoRI sites. The expression

plasmid for t-Darpp was generated by PCR amplification of the full-

length coding sequence of t-Darpp cDNA and cloning into the HindIII and

XhoI sites of pcDNA3.1Zeo. Darpp-32 (T34 ! A or T75 ! A) and t-Darpp(T75 ! A) mutants were produced by site-directed mutagenesis using

QuikChange kit (Stratagene, La Jolla, CA). AGS cells were transfected using

LipofectAMINE (Invitrogen Life Technologies). Stably transfected AGS cells

expressing Darpp-32, t-Darpp, or vector control [empty pCDNA3 (Zeo)]were therefore selected for by addition of 1 mg/mL zeocin (Invitrogen Life

Technologies) to the growth medium. After 3 weeks of selection, several cell

colonies were isolated using cloning rings and then transferred to freshplates containing F-12 (HAM) medium, 10% FBS, and zeocin. These clones

were propagated under selection and the protein expression of Darpp-32

and t-Darpp was determined by Western blot analysis.

Apoptosis assay (terminal deoxynucleotidyl transferase–mediatednick-end labeling). RKO cells were seeded into 8-well chamber slides and

transfected with 400 ng of pcDNA3-based expression plasmids per well, using

Fugene according to the manufacturer’s instructions (Roche Diagnostics,

Indianapolis, IN). After 24 hours, nontreated and camptothecin-treated cellswere stained by terminal deoxynucleotidyl transferase–mediated nick-end

labeling (TUNEL), according to the manufacturer’s instructions (Roche

Diagnostics), and then visualized by FITC (green fluorescence). Darpp-32 and

t-Darpp expression was determined by immunofluorescence staining induplicate wells with Darpp-32 antibody (Santa Cruz Biotechnology) and

donkey anti-rabbit IgG (HL)-rhodamine conjugate (Red; Jackson Immuno-

Research Laboratories, West Grove, PA). Transfection efficiency wasreproducibly f40% for all constructs, and evenly distributed in all wells.

TUNEL-positive cells and Darpp-32- or t-Darpp-expressing cells (20 random

fields at 40�, >400 cells) were counted. The percentage of apoptosis was

determined in Darpp-32- or t-Darpp-expressing cells versus nonexpressingcells after correction for background with vector alone.

Apoptosis assay (Annexin V). AGS stables expressing Darpp-32,

t-Darpp, or empty vector (Zeo) were seeded onto 60-mm culture plates.

After 18 hours, nontreated and drug-treated (camptothecin, sodium butyrate,and ceramide) cells were stained with Annexin V/biotin, streptavidin/APC

(BD Biosciences PharMingen, San Diego, CA), and propidium iodide (PI),

using the Annexin V-Biotin apoptosis detection kit (R&D Systems,Minneapolis, MN). The cells were then resuspended in 1� binding buffer at

a dilution of 106 cells/mL, for subsequent fluorescence-activated cell sorting

(FACS) analysis. For accurate gating of different cell populations, controls

included nontreated AGS-zeo (AGS cells expressing empty vector), ethanol-and PI-treated AGS-zeo, and streptavidin/APC-treated AGS-zeo. Formaximal

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Cancer Res 2005; 65: (15). August 1, 2005 6584 www.aacrjournals.org



signal, FACS analysis was done within 1 hour after cell resuspension. A dot

plot of the X-axis (FL1), being the log of Annexin V fluorescence, and the Y-

axis (FL2), which reflects the PI fluorescence, was obtained by flow cytometry

(Becton Dickinson, Mountain View, CA).Mitochondrial apoptosis assay. This assay, which is based on disruption

of the mitochondrial transmembrane potential (one of the critical events to

occur following induction of apoptosis), was done using the MitoCapture

mitochondrial apoptosis detection kit (BioVision, Mountain View, CA), as

per manufacturer’s instructions. AGS stable cell lines (Darpp-32, t-Darpp,

and empty vector control) were plated in 8-well chamber slides in duplicate

wells. Apoptosis was induced by treating cells with 20 Amol/L camptothecin

for 2 hours. Treated and untreated cells were stained with MitoCapture

reagent and then observed under a fluorescence microscope using a band-

pass filter that permits fluorescence from fluorescein and rhodamine. In

healthy cells, MitoCapture dye accumulated in the mitochondria, giving off a

bright red fluorescence. In apoptotic cells, the dye does not aggregate in the

mitochondria but remains in the cytoplasm and emits a green fluorescence.Luciferase assays. To investigate whether the antiapoptotic effects of

Darpp-32 and t-Darpp involve p53 suppression, luciferase activity assays

using the PG13 reporter plasmid and the p21 reporter plasmid p21-Luc were

done in H1299 cells, a p53 null cell line. The pcDNA3-based mammalian

expression plasmids for Darpp-32 and t-Darpp were described above. The

pcDNA3-Fp53 and pcDNA3-DNp73 were described elsewhere (28). PG13 is a

reporter plasmid containing 13-tandem repeats of the p53 consensus DNA

binding site (29). DNp73 was used as a positive control, as it suppresses the

transcriptional activity of wild-type p53, as previously described (30). H1299

cells were transiently transfected with the constructs, using Fugene (Roche

Diagnostics), and luciferase activity was normalized for Renilla luciferase

activity (Promega, Madison, WI).

Western blot analysis. AGS and H1299 cells were transfected withpcDNA3 expression plasmids for p53, p53 and Darpp-32, p53 and t-Darpp,

p53 and DNp73, Darpp-32, t-Darpp, or empty vector alone. Total cell lysates

were prepared 24 hours post-transfection and subjected to immunoblot

analysis for p21 and Hdm2. Protein lysates were normalized for h-actin

Figure 1. Genomic structure and localization of DARPP-32. A, genomic structure of DARPP-32 and t-DARPP. The mRNA of DARPP-32 is 1,983 bp, including theuntranslated 3V and 5Vends, while t-DARPP is 1,502 bp. DARPP-32 and t-DARPP share identical sequence from exon 2 to the 3V end. Exon 1 of t-DARPP is splicedfrom the intron 1 of DARPP-32. DARPP-32 encodes a protein of 204 amino acids, whereas t-DARPP encodes a 168-amino-acid protein. Darpp-32 contains fourphosphorylation sites at T34, T75, Ser102, and Ser137, whereas t-Darpp lacks the T34 phosphorylation site of Darpp-32. B, genomic localization of DARPP-32 with FISHusing BAC clone CTD-2019C10 (Research Genetics) as a probe for DARPP-32 on a normal metaphase spread. Arrows indicate the green FITC hybridization signals.Right, ideogram of chromosome 17 (left), together with the inverted DAPI banding (middle ) and FISH hybridization signals on the two chromosome 17 (right ). The FISHsignals localize to chromosome band 17q12-q21 indicating the locus for DARPP-32.

Table 1. FISH and immunohistochemical analysis of 257UGCs on tumor tissue arrays

FISH score Immunohistochemistry score Total

0-1 2 3

0-1, n = 176 (%) 75 (43) 55 (31) 46 (26) 176

2, n = 54 (%) 25 (46) 20 (37) 9 (17) 543, n = 12 (%) 2 (17) 4 (33) 6 (50)* 12

4, n = 15 (%) 2 (13) 4 (27) 9 (60)* 15

Total, n = 257 (%) 104 (41) 83 (32) 70 (27) 257

NOTE: FISH score, 0-1 = 2-5 signals, 2 = 6-9 signals, 3 = 10-15 signals,

4 = >15 signals.*P < 0.01.

Darrp-32 and Cancer




content. Rabbit polyclonal antibodies against Darpp-32 (H-62, SC-11365,

Santa Cruz Biotechnology) and phospho-Darpp-32 (T34) and (T75; CellSignaling Technology, Beverly, MA) were obtained. Mouse monoclonal

antibody against HDM2 (IF2) and p21Waf1 (Ab-1, OP64) were obtained

from Oncogene Research Products (La Jolla, CA). Mouse monoclonal anti-

actin antibody (C-2, SC-8432) was obtained from Santa Cruz Biotechnology.Mouse monoclonal anti-Bcl2 antibody (610539) was received from BD

Transduction Laboratories (Lexington, KY).

Darpp-32 and t-Darpp small interfering RNA. Cell lysates from AGS

cells stably expressing empty vector, Darpp-32 or t-Darpp, with and withouttransfection of small interfering RNA (siRNA) oligonucleotides specific for

Darpp-32 and t-Darpp (sc-35173), were subjected to immunoblot analysis

for Darpp-32 and Bcl2. Transfection was done using siRNA transfectionreagent (sc-29528) and Transfection medium (sc-36868), following the

manufacturer’s instructions (Santa Cruz Biotechnology). Lysates were

normalized for equal h-actin loading into the gel.

Results

Genomic structure and localization of DARPP-32. We haverecently cloned DARPP-32 and its truncated isoform t-DARPP fromprimary gastric cancer (6, 7). The genomic structure of DARPP-32

Figure 2. mRNA and protein expression of Darpp-32 in primary UGCs. A-B, quantification of expression of Darpp-32 and t-Darpp in 74 UGCs using an iCycler(Bio-Rad). The expression of Darpp-32 and t-Darpp was compared with Hprt1 gene, which had minimal variation in all normal and neoplastic gastric samples that wetested. Tumor samples were compared to the average expression of 15 normal gastric mucosa samples (SE was <5%). Sample number (horizontal axis) and foldoverexpression (vertical axis). We used an arbitrary cutoff value of 3-fold as a minimum for any overexpression, which is represented by a horizontal line crossing at thisthreshold. There was a strong correlation between Darpp-32 and t-Darpp overexpression (P < 0.001). Seventy percent of the tumors had overexpression of Darpp-32and t-Darpp compared with normal samples (>3-fold). C-E, FISH and immunohistochemistry on tissue array with representative samples of UGCs from the tissuemicroarrays, with a spectrum of glandular differentiation tumor. C, left, FISH using the DARPP-32 probe (CTD-2019C10) and FITC for signal detection. Amplification ofDARPP-32 has occurred, with several signals in each cell from the intestinal-type gastric adenocarcinoma sample. Right, strong immunohistochemistry (3+) withDarpp-32 in a replicate section, as used for FISH (left ). Middle, no immunohistochemical staining for normal mucosa adjacent to the tumor from the same patient as inthe left and right . D, immunohistochemistry on a normal mucosa (left ) and intestinal-type gastroesophageal junctional adenocarcinoma (right), showing strongimmunohistochemical staining in the tumor but not in the normal mucosa from the same patient. E, immunohistochemistry on a normal mucosa (left ) and diffuse-typegastroesophageal junctional adenocarcinoma (right ), showing strong immunohistochemical staining in the tumor but not in the normal mucosa from the same patient.F, example of signet ring cell gastric carcinoma with strong Darpp-32 immunoreactivity (right ). Left, normal mucosa from the same patient. Original magnification, 100�.Inset, both an apparent nuclear and cytoplasmic staining pattern. Original magnification, 1,000�. The abundant intracytoplasmic mucin vacuoles in this variant ofadenocarcinoma show no staining.

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and t-DARPP is depicted in Fig. 1A . The mRNA for DARPP-32 andt-DARPP are 1,983 and 1,502 bp, respectively. DARPP-32 andt-DARPP share sequence identity from exon 2 to the 3V end.However, exon 1 of t-DARPP is spliced from intron 1 of DARPP-32.A protein of 204 amino acids is encoded by DARPP-32, whereast-DARPP encodes a 168-amino-acid protein. Darpp-32 containsfour phosphorylation sites, at T34, T75, S102 and S137, whereast-Darpp lacks the T34 phosphorylation site of Darpp-32. To verifythe chromosomal locus for DARPP-32, we did FISH analysis usingthe BAC clone CTD-2019C10 (Research Genetics) as a probe forDARPP-32 on a normal metaphase spread. A positive signal forDARPP-32 from FISH was indicated by the green FITC hybridiza-tion fluorescence depicted in Fig. 1B (arrows , left). Together withthe inverted DAPI banding and FISH hybridization on the twochromosome 17, the FISH signals were found to localize tochromosome band 17q12-q21 indicating the locus for DARPP-32(Fig. 1B , right).DNA copy numbers, protein, and mRNA levels of DARPP-32

in upper gastrointestinal adenocarcinomas. To evaluate theDNA copy numbers and protein expression of DARPP-32, we didFISH and immunohistochemical analysis on identical replicates ofthe tumor tissue array. FISH and immunohistochemical data were

obtained from 257 tumors and 61 normal samples. Analysis of FISHresults indicated frequent DNA amplification of DARPP-32 inUGCs. Of the tumor samples, 88 (32%) had DNA amplification andshowed more than five signals in at least 50% of tumor cellsanalyzed; 27 (11%) had an average of >10 DARPP-32 signals pertumor cell; whereas 153 (60%) showed moderate-to-strongimmunohistochemical staining for Darpp-32. None of the normalstomach tissue controls showed more than two signals. Immuno-histochemical analysis on tissue arrays showed a moderate tostrong (2+ to 3+) immunostaining for Darpp-32 in 153 tumors(59%). Normal tissue samples had absent to weak (0 to 1+)immunostaining for Darpp-32. This result indicates that over-expression of Darpp-32 is more frequent than its amplificationsuggesting that amplification is not the sole mechanism. High-levelof Darpp-32 protein overexpression (2+ to 3+) was detected in 29cases from 54 (53%) samples with a FISH score of 2, and in 23 casesfrom 27 (85%) samples with a FISH score of 3 to 4 (P < 0.01).Therefore, a strong correlation was found between high-levelamplification of DARPP-32 and its increased protein expressionin UGC tumor tissue arrays. The results and the correlation ofFISH and immunohistochemistry scores in 257 UGCs aresummarized in Table 1, whereas Fig. 2 provides examples of FISH

Figure 3. DARPP-32 counteracts apoptosis induced by camptothecin (CPT ). Darpp-32 and t-Darpp confer camptothecin drug resistance to wild-type p53 harboringtumor cells (RKO). RKO cells were transfected with Darpp-32-pcDNA3.1 (left ) or t-Darpp-pcDNA3.1 (right ) expression plasmids. Twenty-four hours after transfection,cells were treated with 5 Amol/L camptothecin for 18 hours or left untreated (not depicted). After fixation with paraformaldehyde, each well underwent both TUNELstaining in green and immunofluorescence with COOH-terminal Darpp-32-specific polyclonal antibody, which detects both Darpp-32 and t-Darpp (red fluorescence ).DAPI was used as a nuclear counter stain (blue ). Bottom, combined picture (red , green , and blue ) showing that Darpp-32- or t-Darpp-expressing cells (white arrows )are virtually protected from camptothecin-induced apoptosis. The DAPI (blue fluorescence) was used as nuclear counter stain to count all cells. The same experimentwas done using phosphorylation mutants for Darpp-32 and t-Darpp and showed loss of the antiapoptotic potential (not depicted), as summarized in (C ). B, RKO cellstransfected with Darpp-32 and analyzed with TUNEL assay without camptothecin treatment indicate absence of green apoptotic signals, ruling out any effect oftransfection on apoptosis. C, Western blot analysis following transient transfection with wild-type Darpp-32, t-Darpp, and phosphorylation mutants. pcDNA3 expressionplasmids used (top ). Antibodies used for blotting (right ). The blot shows a strong specific band for Darpp-32 (f32 kDa) and t-Darpp (f29 kDa). Antibodies forphosph-T34 or -T75 Darpp-32 show that Darpp-32 had two phosphor bands, pT34 and pT75, whereas t-Darpp shows one phosphor band for pT75. The phosphorylationmutants do not show phosphorylation signal at the mutated site. h-Actin was used for normalization for equal loading. D, summary of the percentage of apoptosisin Darpp-32- and t-Darpp-expressing cells compared with pCDNA3-transfected cells, with and without 5 Amol/L camptothecin treatment for 18 hours. Results frommutants are shown and indicate loss of the antiapoptotic potential. The experiments were repeated three times. Columns, means; bars, FSE.

Darrp-32 and Cancer




and immunohistochemistry in UGCs. In addition, we haveobserved that protein overexpression of Darpp-32 is more frequentin diffuse-type rather than intestinal-type UGC (P = 0.05).To investigate whether DARPP-32 gene amplification causes

commensurate mRNA gene expression, we set out to analyze thelevels of mRNA expression. The expression of DARPP-32 andt-DARPP was compared with that of the HPRT1 gene, for whichminimal variation was seen in all the normal and neoplastic tumorsamples tested. The expression in each tumor sample wascompared with the expression in 15 normal gastric mucosasamples (FSE <5%). We used an arbitrary cutoff value of a 3-foldincrease as the minimum considered to indicate overexpression.Overexpression was seen in 50 tumors (68%), along withconcomitant overexpression of Darpp-32 and t-Darpp (P < 0.001;Fig. 2A and B). Therefore, the mRNA overexpression frequency isvery close to the observed protein overexpression followingimmunohistochemical staining and is also higher than genomicamplification. This finding indicates that in addition to DNAamplification, transcriptional and/or posttranscriptional mecha-nism may play a role for Darpp-32 overexpression. FISH and mRNAdata were available for 38 tumors. Tumors that had an average ofmore than five signals per cell (score z 2) had a higher average foldmRNA expression for Darpp-32 or t-Darpp (>50-fold) comparedwith tumors without amplification (<30-fold). The data aresummarized in Table 1 and Fig. 2.Darpp-32 counteracts apoptosis induced by camptothecin.

The antiapoptotic activity of Darpp-32 and t-Darpp was investi-gated in RKO cells that express wild-type p53. The results, following18 hours of treatment with camptothecin, revealed a dramatic4-fold reduction in apoptotic cell count in Darpp-32- or t-Darpp-expressing cells compared with control (Fig. 3A). The RKO cellstransfected with Darpp-32 but not treated with camptothecin didnot show any green fluorescence in the TUNEL assay, ruling outany effect of transfection on apoptosis (Fig. 3B). Western blotanalysis following transient transfection with expression plasmidsfor Darpp-32, t-Darpp, or the T34 and T75 mutants confirmed the

strong expression of these proteins and the lack of phosphorylationsignal that correlated with the mutants tested (Fig. 3C). The sameTUNEL experiment was done using phosphorylation mutants forDarpp-32 (T34 ! A or T75 ! A) and t-Darpp (T75 ! A). For thesevariants, a complete loss of antiapoptotic protection againstcamptothecin was observed (Fig. 3D).Darpp-32 counters apoptosis induced by different drugs. To

further investigate the antiapoptotic effect of DARPP-32, severaldrugs were tested using Annexin V apoptosis assay. Overall, theresults obtained for camptothecin, ceramide, and sodium butyratein the Annexin V assay confirmed the strong antiapoptotic effectfor Darpp-32 and t-Darpp observed in the TUNEL assay. Thepercentages of early apoptotic cells, Annexin V positive and PInegative, are summarized in Fig. 4 (bottom). These results indicatethat Darpp-32 and t-Darpp provide an antiapoptotic advantage forgastrointestinal cells against several chemotherapeutic drugs thatinduce apoptosis through different mechanisms. However, thet-Darpp-expressing AGS cells were considerably more resistant toapoptosis than Darpp-32, following ceramide treatment.Darpp-32 preserves mitochondrial transmembrane

potential. In healthy cells, the MitoCapture dye accumulated inthe mitochondria in a similar manner to untreated cells (Fig. 5,left); the same was seen in Darpp-32 and t-Darpp camptothecin-treated cells (Fig. 5, right). In apoptotic cells, the dye did notaggregate in the mitochondria, as seen following camptothecintreatment of cells transfected with empty vector (Zeo) cells (Fig. 5,top right). This indicates that both Darpp-32 and t-Darpp protectedthe mitochondrial transmembrane potential in AGS cells followingtreatment with camptothecin.Darpp-32 and t-Darpp abrogate apoptosis in a p53-

independent mechanism. As expected, cotransfection of DNp73and p53 dramatically suppressed the transcriptional activity ofwild-type p53, as measured by PG13-luc and p21-luc reporters. Incontrast, cotransfection of p53 with either Darpp-32 or t-Darpp didnot suppress the p53 activity (Fig. 6A and B). Western blot analysisshows that transfection of Darpp-32 or t-Darpp does not change

Figure 4. DARPP-32 counteracts apoptosis induced bydifferent drugs. Darpp-32 and t-Darpp confer drug resistance tocamptothecin, acetyl ceramide, and sodium butyrate in tumorcells (AGS). Stable AGS cell lines were generated forDarpp-32, t-Darpp, and empty vector using pcDNA3.1expression plasmids. Top, results after treatment of thesecell lines for 24 hours with 20 Amol/L camptothecin (CPT),20 Amol/L ceramide, or 5 mmol/L butyrate followed by AnnexinV apoptosis assay. Cells were stained with Annexin V/biotinand streptavidin/APC (BD Biosciences PharMingen) and PIusing the Annexin V-Biotin apoptosis detection kit (R&DSystems). The dot plot of the X -axis (FL1) of the log of AnnexinV fluorescence and the Y -axis (FL2), which reflects the PIfluorescence, was obtained by flow cytometry (BectonDickinson). The early apoptotic cells, typically Annexin Vpositive and PI negative (bottom right quadrant ). Summary ofthe data (bottom ) showing percentage of early apoptotic cellsin each analysis.

Cancer Research




the base levels of both Hdm2 and p21 , p53 target genes (Fig. 6C). Inaddition, cotransfection of p53 with Darpp-32 or t-Darpp did notalter the levels of Hdm2 or p21, whereas cotransfection of p53 withDNp73 dramatically decreased the levels of both Hdm2 and p21compared with p53 alone (Fig. 6C). Taken together, these data showthat neither Darpp-32 nor t-Darpp expression inhibited the p53transcriptional function, which is the basis of the p53 proapoptoticactivity. Moreover, they confirmed our finding that the inhibitoryeffect of Darpp-32 and t-Darpp on apoptosis involves a mechanismdistinct from p53 suppression.Darpp-32 and t-Darpp up-regulate Bcl2. Because Darpp-32

and t-Darpp are protective against disruption of the mitochondrialtransmembrane potential following induction of apoptosis withcamptothecin (Fig. 5), we postulated that Bcl2 could potentially beinvolved in this specific antiapoptotic mechanism. The Darpp-32and t-Darpp AGS stable cells expressed high levels of Bcl2 that werenot detected in the vector control cells (Fig. 6D). Similar resultswere obtained upon transient transfections. On the other hand,transfection with a siRNA oligonucleotide that inhibits Darpp-32and t-Darpp led to almost complete loss of Bcl2 expression in theDarpp-32 and t-Darpp stable cell lines (Fig. 6D). The expression ofBcl2 was therefore up-regulated by both Darpp-32 and t-Darpp inAGS cells.

Discussion

We have earlier cloned Darpp-32 and a novel truncated isoform,t-Darpp, in gastric cancer (7). In this study, we verified the

cytogenetic localization of the gene encoding Darpp-32 tochromosome band 17q12-q21. Our study shows that overexpressionof Darpp-32, at the mRNA and protein level, in UGCs is morefrequent than genomic DNA amplification. Using tumor tissuearrays, we detected gene amplification of DARPP-32 in approxi-mately one third of UGCs, whereas overexpression of Darpp-32 atthe mRNA and protein levels was detected in more than two thirdsof tumors. Therefore, Darpp-32 overexpression was not limited togenomic amplification. The similar high frequency of Darpp-32overexpression at both mRNA and protein level suggests thattranscriptional and/or posttranscriptional regulatory mechanismsmay be involved. Nevertheless, tumor samples with a high level ofgenomic DARPP-32 DNA amplification (>10 signals per cell)showed consistently higher immunohistochemical staining (3+)than any other tumor (P = 0.01). This indicates that amplification,although not the only mechanism, can play a role in inducing high-levels of Darpp-32 expression. Interestingly, protein overexpressionof Darpp-32 was more frequent in diffuse-type UGC (P = 0.05).Diffuse-type tumors are known to be more aggressive with a pooroutcome (1).Interestingly, we have detected concomitant mRNA overexpres-

sion of Darpp-32 and t-Darpp (P < 0.001). The Darpp-32 and t-Darpp were frequently overexpressed in both distal and proximalgastroesophageal adenocarcinomas. We have observed a similartrend for Darpp-32 and t-Darpp overexpression in a small numberof common adenocarcinomas, including breast, colon, lung, andprostate (8). However, a recent study has shown that esophagealsquamous cell carcinomas frequently overexpress Darpp-32 but nott-Darpp (9). In a related study, amplification and overexpression ofERBB2 , which coincides at the same locus as DARPP-32 (17q12q21),was found in f10% of both diffuse-type and intestinal-type gastriccancers (31). The frequent gene amplification and high mRNA andprotein expression levels of Darpp-32 suggest that it plays animportant role in tumor development and progression of UGCs.Darpp-32 is known as a neurosignaling molecule that plays a

crucial role in dopamine signaling in the brain (32, 33). To date, thebiological function of Darpp-32 in normal tissues and in cancerremains unknown. In this study, we have shown that Darpp-32 andt-Darpp proteins have a potential antiapoptotic role and providedrug resistance with a survival advantage to neoplastic gastroin-testinal cells. Apoptosis, or programmed cell death, is an essentialphysiologic process that is involved in the elimination of infected,damaged, or unwanted cells (34–37). Deregulation of apoptosis isan important step in cancer development, with aberrant cancercells escaping cell death and acquiring additional neoplasticfeatures. This feature renders several cancers, including UGCs,resistant to cancer therapy (25, 38, 39). These independent methods(i.e., TUNEL, Annexin V, and mitochondrial potential assays)revealed a consistent dramatic reduction in apoptosis in cellsexpressing either Darpp-32 or t-Darpp. Phosphorylation mutants ofDarpp-32 (T34 ! A or T75 ! A) and t-Darpp (T75 ! A) did notconfer any antiapoptotic protection against camptothecin. Thisfinding shows that the T34 and T75 phosphorylation sites may becritical to maintain the full antiapoptotic potential of both Darpp-32 and t-Darpp. The antiapoptotic advantage was observed ingastric cancer cells expressing either Darpp-32 or t-Darpp followingtreatment with camptothecin, ceramide, or sodium butyrate.However, following induction of apoptosis with ceramide, weobserved that the t-Darpp-expressing cells were considerably moreresistant to apoptosis than Darpp-32 expressing cells. This may bea drug-specific effect, because Darpp-32 and t-Darpp conferred

Figure 5. Darpp-32 preserves mitochondrial transmemberane potential. AGSstably transfected cell lines for empty vector (Zeo), Darpp-32, or t-Darpp werestained with MitoCapture mitochondrial apoptosis detection kit (BioVision).AGS cells treated with 5 Amol/L camptothecin for 2 hours (right ) or nontreatedAGS cells (left ) were analyzed using an Olympus fluorescent microscope. Inhealthy cells, the MitoCapture dye accumulates in the mitochondria with a brightred fluorescence, as shown in all nontreated AGS cells, as well as in treatedDarpp-32 and t-Darpp expressing AGS cells. In apoptotic cells, the dye does notaggregate in the mitochondria. This was seen as absence of red fluorescence, asshown following camptothecin treatment in empty vector (Zeo)–expressing cells.

Darrp-32 and Cancer




similar drug resistance against both camptothecin and sodiumbutyrate. It has to be noted that Darpp-32 has two mainphosphorylation sites (T34 and T75), whereas t-Darpp lacks theT34 (40); therefore, differences can be expected.These drugs are known to act upon different signaling

pathways. Ceramide activates protein phosphatases such as PP1and PP2A, through which it can indirectly inhibit pro-growthsignaling kinases such as PKC isoforms and Akt (41). On theother hand, camptothecin is a well-established cancer drug thatinhibits topoisomerase I and blocks DNA replication in neoplasticcells (42). Camptothecin treatment results in up-regulation andactivation of p53 and down-regulation of Bcl2 and Bcl-xl (43, 44).The sodium butyrate is a histone deacetylase inhibitor thatinduces the loss of the mitochondrial transmembrane potentialand the release of cytochrome c into the cytoplasm, as well asactivating both caspase 9 and caspase 3 (45). Sodium butyratecan also induce apoptosis in a p53-independent mechanism

through the Fas signaling pathway (46). Our results that Darpp-32and t-Darpp can provide resistance to these different drugs andact upon different signaling cascades suggest that Darpp-32proteins may be related to the drug resistance phenotype inUGCs.Several proteins provide protection against apoptosis through

inhibition of 53-induced apoptosis. Therefore, we sought toinvestigate whether Darpp-32 proteins can interfere with the p53signaling mechanism. Upon cellular stress, p53/p21 becomesactivated and up-regulates the transcription of a number of keyproapoptotic genes (NOXA , PUMA , BAX , etc.), that in turn tocarry out apoptotic signal downstream to the apoptoticmachinery. Interestingly, using reporter analysis and Westernblotting, we showed that expression of Darpp-32 or t-Darpp failedto suppress the transcriptional activity of p53. The controlexperiment using dnp73 showed, as expected, a strong inhibitionof p53. Thus, we concluded that the antiapoptotic effect of

Figure 6. DARPP-32 counteracts apoptosis in a p53-independent mechanism. A-B, luciferase activity using the PG13 and p21 promoter luciferase constructs in p53null H1299 cells. DNp73 was used as a positive control that mediates suppression of the wild-type p53/TAp73-responsive reporter construct PG13-luciferase. Luciferaseactivity is normalized for Renilla luciferase activity. Coexpressed DNp73 caused a dramatic suppression of the transcriptional activity of wild-type p53 and p21. Thecotransfection of p53 with Darpp-32 or t-Darpp did not suppress the p53 or the p21 activity. Columns, averages of three independent experiments; bars, FSE.C, immunoblots of H1299 cells transfected with p53 or DNp73, either alone or in combination with Darpp-32 or t-Darpp. Transfections were done in parallel with (A-B ).Samples were normalized to h-actin before loading. The transfection of empty vector alone shows low base levels of Hdm2 and p21, which remained similar aftertransfection of Darpp-32 or t-Darpp alone. The transfection of p53 induced transactivation of the endogenous target genes p21 and Hdm2 (lane 2). Cotransfection ofp53 with Darpp-32 or t-Darpp did not change the levels of Hdm2 or p21, whereas cotransfection of p53 with DNp73 significantly reduced the levels of Hdm2 and p21compared with p53 alone. D, immunoblots of AGS cells stably expressing empty vector (Zeo), Darpp-32, or t-Darpp, with and without transfection of siRNAoligonucleotide specific for Darpp-32 (Santa Cruz Biotechnology). Samples were normalized to h-actin before loading. The Darpp-32 and t-Darpp cells show high levelsof Bcl2 that is not detected in the vector control. Transfection with a siRNA oligonucleotide leads to complete loss of the Bcl2 in AGS cells stably expressing eitherDarpp-32 or t-Darpp.

Cancer Research




Darpp-32 and t-Darpp was conferred through a p53-independentmechanism. These findings may also explain that apoptosis bythree different anticancer drugs was countered by Darpp-32 andt-Darpp.The preservation of the mitochondrial transmembrane poten-

tial by Darpp-32 or t-Darpp following induction of apoptosis withcamptothecin, ascertained independently the strong antiapoptoticpotential of Darpp-32 and t-Darpp. Disruption of the mitochon-drial transmembrane potential is one of the early events to occurfollowing induction of apoptosis (47–49). Taken together, theseresults suggested that the antiapoptotic mechanism employed byDarpp-32 and t-Darpp may involve a mitochondrial-relatedmolecule downstream of p53. In this report, we showed thatexpression of Bcl2 was up-regulated by Darpp-32 and t-Darppexpression. Expression knockdown of Darpp-32 or t-Darpp bysiRNA confirmed that the levels of Bcl2 are specifically regulatedby Darpp-32 and t-Darpp. Bcl2 inhibits drug-induced apoptosis bypreventing cytochrome c release (50) and subsequent abrogationof the activation of caspases (51–54). The interactions betweenantiapoptotic Bcl2 family members (such as Bcl-XL and Mcl1) andproapoptotic proteins (such as Bad and Bax) determine whetherapoptosis occurs (55). Thus, we have identified a novel

antiapoptotic signaling pathway by which Darpp-32 proteinscounter apoptosis.In conclusion, our results indicate that tumor samples with

high-level DNA amplification have consistently high immunohis-tochemical staining for Darpp-32. However, mRNA and proteinoverexpression of Darpp-32 were more frequent than genomic DNAamplification in UGCs. Our findings underscore both a biologicalfunction and a potential oncogenic role for Darpp-32 and t-Darppin UGCs. Overexpression of Darpp-32 and t-Darpp is associatedwith a potent antiapoptotic advantage against several knowndrugs. This pathway involves both Bcl2 up-regulation andpreservation of the mitochondrial transmembrane potential in ap53-independent manner. Further studies are ongoing to under-stand the signaling mechanism of Darpp-32 proteins in cancer.

Acknowledgments

Received 4/26/2005; revised 5/13/2005; accepted 5/24/2005.Grant support: National Cancer Institute grant R01CA93999 (W. El-Rifai).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Asta Varis and Sarah de la Rue for their assistance.

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Cancer Research




2005;65:6583-6592. Cancer Res Abbes Belkhiri, Alexander Zaika, Nataliya Pidkovka, et al. Gastrointestinal Carcinomas

: a Novel Antiapoptotic Gene in UpperDarpp-32

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Darpp-32: a Novel Antiapoptotic Gene in Upper Gastrointestinal … · truncated isoform t-DARPP (AY070271) from adenocarcinoma of the stomach with 17q amplicon (6, 7). In addition,