DNA AND CELL BIOLOGYVolume 25, Number 3, 2006© Mary Ann Liebert, Inc.Pp. 135–141

Regulation of Apoptosis by a Prostate-Specific and Prostate Cancer-Associated Noncoding Gene, PCGEM1

XIAOQIN FU, LAKSHMI RAVINDRANATH, NICHOLAS TRAN, GYORGY PETROVICS, and SHIV SRIVASTAVA

ABSTRACT

PCGEM1 is a prostate tissue-specific, and prostate cancer-associated noncoding RNA (ncRNA) gene. Previ-ous results revealed a significant association of elevated PCGEM1 expression levels in prostate cancer cells ofAfrican–American patients, whose mortality rate is the highest among prostate cancer patients. Functionalstudy of PCGEM1 demonstrated a marked increase in colony formation in LNCaP prostate cancer cells andNIH3T3 mouse fibroblast cells. This study demonstrates that PCGEM1 overexpression in LNCaP cell culturemodel results in the inhibition of apoptosis induced by doxorubicin (DOX). Induction of p53 and p21Waf1/Cip1

by DOX were delayed in LNCaP cells stably overexpressing PCGEM1 (LNCaP-PCGEM1 cells) compared tocontrol LNCaP cells. The protein levels of cleaved caspase 7, and cleaved PARP were attenuated in DOX-treated LNCaP-PCGEM1 cells compared to control LNCaP cells. Similar results were observed in LNCaPcells transiently overexpressing PCGEM1. The inhibition of PARP cleavage by PCGEM1 overexpression wasalso observed in LNCaP-PCGEM1 cells incubated with etoposide and sodium selenite. Fluorescence-ActivatedCell Sorter Annexin-V analysis revealed significantly lower percentage of apoptotic cells in DOX-treatedLNCaP-PCGEM1 cells compared to control LNCaP cells. The attenuation of apoptic response appears to beandrogen dependent in this experimental model, as androgen-independent variants of LNCaP cells did notexhibit this response. In summary, this study provides new insights into cell biologic functions and novel fea-tures of an ncRNA. Further, these data unravel biological mechanisms of cell growth/cell survival-associatedfunctions of this ncRNA in a widely used prostate cancer cell culture model.

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INTRODUCTION

PCGEM1 WAS ORIGINALLY DISCOVERED in our laboratory asa novel cDNA sequence showing prostate cancer-associ-

ated overexpression in a global gene expression screen ofprostate cancer-associated gene expression alterations (Srikan-tan et al., 2000). One of the unique characteristics of PCGEM1is its prostate tissue-specific expression restricted to glandularepithelial cells. Evaluation of PCGEM1 expression revealedsignificantly elevated PCGEM1 expression levels in prostatecancer cells of African–American patients, whose mortality rateis the highest among prostate cancer patients, suggesting po-tential roles for PCGEM1 in prostate cancer biology (Petrovicset al., 2004). PCGEM1 overexpression in LNCaP and inNIH3T3 cells promotes cell proliferation and a dramatic in-crease in colony formation, suggesting for a biological role of

PCGEM1 in cell growth regulation (Petrovics et al., 2004). In-creased phosphorylation of RB (Ser807/811) was observed inLNCaP and NIH3T3 cells overexpressing PCGEM1 (Petrovicset al., 2004), suggesting for a role of PCGEM1 in regulation ofcell cycle.

PCGEM1 is expressed as noncoding poly(A)� RNA of 1643nucleotides. PCGEM1 along with PCA3 (DD3) (Bussemakerset al., 1999) represent a novel class of prostate-specific geneswhose functions remain to be defined in prostate biology andcancer. Diagnostic utility of PCA3 (Landers et al., 2005) inprostate cancer and association of the PCGEM1 expression inhigh-risk prostate cancer patients have been emphasized. It isonly recently recognized that there is a significantly larger pro-portion of the human genome transcribed as mature cytoplas-mic poly(A)� RNA than previously considered (Huttenhoferet al., 2001; Kapranov et al., 2002; Cawley et al., 2004; Kampa

Department of Surgery, Center for Prostate Disease Research (CPDR), U.S. Military Cancer Institute, Uniformed Services University of theHealth Sciences, Bethesda, Maryland.

et al., 2004). Some of these RNA transcripts include XIST(Brockdorff et al., 1992), H19 (Brannan et al., 1990; Ariel etal., 1997; Ayesh et al., 2002), gadd7/adapt15 (Hollander etal., 1996), His-1 (Li et al., 1997), Bic (Haasch et al., 2002),NTT (Liu et al., 1997), hsr-omega (Lakhotia and Sharma,1996), CR20 (Teramoto et al., 1996), and MALAT-1 (Ji et al.,2003). Analyses of these transcripts suggest functions in geneexpression silencing (XIST, H19), in stress response(gadd7/adapt15, hsr-omega), and in tumorigenesis (H19, His-1, Bic, MALAT-1). NcRNA might exert its functions throughassociation with proteins. For example, BRCA1, a breast andovarian tumor suppressor, is found colocalized with XISTRNA, and supports XIST RNA concentration on the inactiveX chromosome (Ganesan et al., 2002, 2004). Some of thesencRNA are developmentally regulated or show highly re-stricted pattern of gene expression. Overall, our understandingof ncRNAs is still very limited, functional analysis of ncRNAshas promise to define biological functions of this new class ofgenes.

In this study, we report that overexpression of PCGEM1 re-sults in attenuated induction of p53 and p21Waf1/Cip1 by DOXincubation, and resistance to apoptosis in LNCaP prostate can-cer cells but not in androgen-independent variants of LNCaP.This report provides new insights into functions of PCGEM1,a prostate cancer-associated ncRNA gene.

MATERIALS AND METHODS

Cell culture and treatments

LNCaP cells were obtained from American Type Culture Col-lection (ATCC, Rockville, MD) and maintained as recom-mended by the supplier. Cell culture reagents were purchasedfrom Invitrogen Life Technologies, Inc. (Grand Island, NY).DOX, sodium selenite, etoposide, and staurosporine were pur-chased from Sigma Chemical Co. (St. Louis, MO). LNCaP cellswere cultured in RPMI 1640 medium containing 10% (vol/vol)fetal bovine serum at 37°C in a humidified atmosphere of 95%air and 5% CO2. LNCaP cells stably transfected with PCGEM1expression vector (Petrovics et al., 2004) were maintained in thesame culture medium containing puromycin (1 �g/ml). Mediumwas replenished every 3 days, and subcultures were preparedevery 1–2 weeks depending on the growth rates of the cells.

Transient transfection in prostate cancer cells

LNCaP cells (2 � 106) were transfected with 4 �g vector us-ing Nucleofector (Amaxa Biosystems, Gaithersburg, MD) ac-cording to the manufacturer’s protocol. LNCaP transfectantswere then cultured in poly-L-lysine coated six-well plates(Greiner Bio-one, Monroe, NC).

RNA isolation and Northern blot analysis of PCGEM1 expression

Total RNAs were extracted using RNA-Bee Reagent (TEL-TEST, Friendswood, TX) according to the manufacturer’s pro-tocol. Buffers (10 �SSC and 10% SDS) were purchased fromQuality Biological Inc. (Gaithersburg, MD). Total RNA (10 �g)from each sample were electrophoresed through 1% agarosegels in 1� NorthernMax®-Gly Gel Prep/Running Buffer (Am-bion, TX), and transferred to 0.2-�m pore-size Nylon transfermembranes (Schleicher & Schuell BioScience, Keene, NH).

A [32P]dCTP-labeled PCGEM1 cDNA probe (#AF223389,

FU ET AL.136

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FIG. 1. PCGEM1 overexpression in LNCaP-PCGEM1 cells.Northern blot analysis of PCGEM1 expression in the threeLNCaP cells isolated from stable transfection of PCGEM1cDNA (lanes 1–3), and in LNCaP cells transfected with vec-tors (lane 4).

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FIG. 2. Effects of PCGEM1 over-expression on p21Waf1/Cip1 and p53. (A)p21Waf1/Cip1 protein levels were analyzedby Western blot in control LNCaP cells(without PCGEM1 overexpression) andLNCaP-PCGEM1 cells (with PCGEM1overexpression). (B) Analysis of the pro-tein levels of p53 and p21Waf1/Cip1 in con-trol LNCaP cells and LNCaP–PCGEM1cells with/without (�) DOX treatment fordifferent lengths of time (4, 8, and 24 h).Tubulin served as an internal control.Quantification of relative band intensity ofp53 (C) and p21Waf1/Cip1 (D) were nor-malized to the intensity of tubulin bandsusing Quantity One (version 4.5.0) com-puter program. Values represent means �SEM (n � 3 independent experiments).Asterisks indicate significant difference(p � 0.05) from control LNCaP cells.

625 to 841 bp) was generated using Prime-It® II Random PrimerLabeling Kit (Stratagene, La Jolla, CA). Hybridization withPCGEM1 cDNA probe was performed at 43°C overnight. Themembranes were washed under the following conditions: 2 �SSC, 0.1% SDS, room temperature, 30 min; 0.2 � SSC, 0.1%SDS, 43°C, 30 min. Filters were exposed to films for 24 h, andthe bands were visualized by autoradiography.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)and Western blot analysis

Cells were lysed in T-PER tissue Protein Extraction Reagent(Pierce, Rockford, IL) containing protease inhibitors (Pierce)followed by sonication (15-sec pulse at a power output of 2 us-ing the VirSonic 100, SP Industries Company, Warminster, PA).Protein concentrations were determined by BCA protein assay(Pierce). Proteins were resolved with SDS-PAGE utilizing 4–12% NuPage Bis-Tris gels and transferred to nitrocellulosemembranes; 4–12% NuPage Bis-Tris Gels, NuPAGE® MESSDS Running Buffer, NuPAGE® Transfer Buffer, and 0.2-�mpore-size nitrocellulose membrane were purchased from Invit-rogen. Nitrocellulose membranes were blocked in 5% nonfatmilk in phosphate-buffered saline (PBS, Quality Biological Inc.,Gaithersburg, MD). Membranes were then incubated with pri-mary antibodies overnight. After washing in PBS containing0.05% (v/v) Tween 20 (PBST), membranes were incubated withhorseradish peroxidase-conjugated secondary antibodies (CellSignaling Technology, Beverly, MA). After washing, membraneswere developed with the Supersignal West Pico Chemilumi-nescent Substrate (PIERCE, Rockford, IL). Antibodies forp21Waf1/Cip1 (# 2946), cleaved caspase 3 (# 9915), cleaved caspase7 (# 9915), cleaved caspase 9 (# 9915), PARP (# 9542) and cleavedPARP (# 9541S) were obtained from Cell Signaling Technology(Beverly, MA). Antibody for p53 (Pab 1801, #sc-98) was obtainedfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Determination of apoptotic cell death by fluorescence-activated cell sorter (FACS)

Apoptosis was evaluated with an EPICS ELITE ESP (Beck-man Coulter, Miami, FL) flow cytometer, using the Annexin-V-PE and 7-AAD staining methods (BD Pharmingen, CA) ac-cording to the manufacturer’s protocol. Briefly, cells werecollected after treatments and washed in ice-cold PBS. Cellswere then resuspended in 100 �l incubation buffer containing5 �l Annexin V and 5 �l 7-AAD staining reagents at a con-centration of 1 � 106 cells, and incubated in the dark for 15min at room temperature before FACS.

RESULTS

PCGEM1 attenuates the inductions of p53 andp21Waf1/Cip1 by doxorubicin

In this study, results of LNCaP cells stably overexpressingPCGEM1 (LNCaP-PCGEM1) were obtained from three inde-pendent colonies of LNCaP-PCGEM1 cells isolated from sta-ble transfection of PCGEM1 cDNA in LNCaP cells. Overex-pressions of PCGEM1 in LNCaP cells were confirmed byNorthern blot analysis (Fig. 1).

Since PCGEM1 expression LNCaP cells showed increasedcell proliferation rate as well as increased RB phosphorylation,we examined cell growth regulatory pathways involving RB(Cdk, p16, p21, and p27) in LNCaP cells harboring ectopic ex-pression of PCGEM1. Decreased expression of p21Waf1/Cip1 wasnoted in LNCAP-PCGEM1 cells compared to control cells (Fig.2A). Since p21Waf1/Cip1 is a direct downstream target of the p53 tumor suppressor gene (Dulic et al., 1994; El-Deiry et al.,1994). We investigated whether the activation of p53 and theinduction of p21Waf1/Cip1 by p53 were affected by PCGEM1overexpression in LNCaP cells. The levels of p53 andp21Waf1/Cip1 proteins were compared in control LNCaP cellsand LNCaP-PCGEM1 cells after incubation with DOX, whichis known to induce p53 as well as p21. Expression of p53 wasbarely detected in both control LNCaP cells and LNCaP-PCGEM1 cells without DOX treatment (Fig. 2B). p53 expres-sion was induced in LNCaP-PCGEM1 cells after DOX treat-ment for 4 h. However, the DOX induced protein levels of p53were higher in control LNCaP cells compared to those inLNCAP-PCGEM1 cells (Fig. 2B). Quantitative evaluations ofp21 and p53 were performed in LNCaP and LNCaP-PCGEM1cells treated with DOX for different time periods. The kinetics

REGULATION OF APOPTOSIS BY PCGEM1 137

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FIG. 3. PCGEM1 inhibits the induction of cleaved caspase 7and cleaved PARP. (A,B) Analysis of cleaved caspase 7 (A)and cleaved PARP (B) by Western blot in control LNCaP cellsand LNCaP-PCGEM1 cells with/without (�) DOX treatmentsfor different lengths of time (4, 8, and 24 h). (C) The proteinlevels of cleaved PARP detected by Western blot in controlLNCaP cells and LNCaP-PCGEM1 cells treated with/without(�) sodium selenite (2 �M) for 72 h. (D) Analysis of cleavedPARP by Western blot in control LNCaP cells and LNCaP-PCGEM1 cells with/without etoposide treatments (2 and 10�M) for 5 days. The expression of tubulin served as an inter-nal control of equal loading. The results are representatives ofthree independent experiments.

of induction of p53 was delayed in LNCaP-PCGEM1 cells com-pared to control LNCaP cells (Fig. 2C), and the levels of p53in both LNCaP and LNCaP-PCGEM1 cells decreased after 24h DOX incubation. Although the induction of p21Waf1/Cip1

protein was observed in both control LNCaP and LNCaP-PCGEM1 cells after 8 h DOX incubation, the protein levels ofp21Waf1/Cip1 in LNCaP-PCGEM1 cells were significantly lowercompared to control LNCaP cells (Fig. 2D). These resultsdemonstrate that the induction of p53 and p21Waf1/Cip1 by DOXis attenuated by PCGEM1 overexpression in LNCaP cells.

Inhibition of apoptosis by PCGEM1 overexpression

The induction of apoptosis by DOX was investigated in con-trol LNCaP cells and in LNCaP-PCGEM1 cells. ControlLNCaP cells and LNCaP-PCGEM1 cells were treated with/without DOX for 4, 8, and 24 h. The expression of cleaved cas-pase 3, 7, and 9 were detected by Western blot analysis. Theexpression of cleaved caspase 3 and cleaved caspase 9 were notdetected in control LNCaP cells or LNCaP-PCGEM1 cells (datanot shown). Cleaved caspase 7 was detected after 8 h DOXtreatment in control LNCaP cells, which further increased af-ter 24 h DOX treatment (Fig. 3A). However, cleaved caspase7 in LNCAP-PCGEM1 cells was not detected even after 24 hDOX treatment (Fig. 3A). This data suggests that overexpres-sion of PCGEM1 attenuated the induction of apoptosis in

LNCaP cells. To further confirm this finding, the expression ofcleaved PARP, a downstream target of caspases, was analyzed.The protein levels of cleaved PARP (89 kDa) the active formof PARP increased after an 8 h DOX treatment in controlLNCaP cells, and further increased after a 24 h DOX treatment(Fig. 3B). In contrast, the expression of cleaved PARP inLNCAP-PCGEM1 cells was only detected after 24 h DOXtreatment, and the protein level was lower compared to controlLNCaP (Fig. 3B). The inhibition of PARP cleavage byPCGEM1 overexpression was also observed in LNCaP-PCGEM1 cells treated with sodium selenite (Fig. 3C) andetoposide (Fig. 3D). These reagents were previously reportedto induce apoptosis in LNCaP cells (Zhong and Oberley, 2001;Salido et al., 2004). The results are representatives of three in-dependent experiments.

Apoptosis was also determined by flow cytometry using An-nexin V-PE/7-AAD staining method (BD Pharmingen). Cellswere collected after 0-h, 24-h, and 48-h incubation with/with-out DOX. Cells were evaluated based on staining for AnnexinV (apoptotic cells) and propidium iodide (necrotic cells). Thepercentages of apoptotic cells in LNCaP-PCGEM1 cells weresignificantly lower compared to control LNCaP cells after 24and 48 h DOX treatments (P � 0.05, n � 3 independent ex-periments) (Fig. 4B).

Effects of PCGEM1 overexpression on apoptosis were alsoexamined in LNCaP cells transiently transfected with PCGEM1

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FIG. 4. Determination of apoptosis in LNCaP-PCGEM1 cells with FACS. Staining of Annexin-V was measured in controlLNCaP cells and LNCaP-PCGEM1 cells after 0 h (A), 24 h (B), and 48 h (C) DOX treatments. (D) The percentages of inducedapoptotic cells in control LNCaP cells (�) and LNCaP-PCGEM1 cells (�) were calculated after DOX incubation for 24 and 48h. Data represents the means and SEM derived from three independent experiments. Asterisks indicate significant difference (p �0.05) from control LNCaP cells with same treatment.

cDNA (constructed into vector pEAK8: pEAK8-PCGEM1 vec-tors) (Fig. 5). LNCaP cells were transfected with pMAX-GFP,pEAK8, or pEAK8-PCGEM1 vectors, respectively, using Nu-cleofector (Amaxa Biosystems). The transfection of pMAX-GFP and expression of GFP served as controls in estimatingtransfection efficiency (�70% after 24 h, data not shown). Af-ter incubation for 48 h, the cells were treated with/without DOX(0.2 �g/ml) for 24 h. LNCaP cells transiently expressingPCGEM1 were evaluated for PARP cleavage (Fig. 5). Com-pared to LNCaP cells with no DOX treatment, the protein lev-els of cleaved PARP were significantly increased in LNCaP-PCGEM1 cells treated with DOX (Fig. 5E). These results arein agreement with data from stably transfected LNCaP-PCGEM1 cells. PCGEM1 effects on apoptosis in LNCaP cells,an androgen dependent CaP cell line, were not observed in an-drogen independent derivatives of LNCaP, C4-2, and C4-2b(Fig. 5C and D).

DISCUSSION

This study demonstrates that overexpression of PCGEM1, aprostate-specific ncRNA gene, attenuates DOX induced ex-pression of p53 and p21Waf1/Cip1, and inhibits apoptosis inLNCaP cells. These results established a novel function ofPCGEM1 in addition to previously described cell growth pro-moting function(s) (Petrovics et al., 2004).

The initial investigations of the role of PCGEM1 in cancerbiology were prompted by our original observations showingPCGEM1 overexpression in prostate cancer cells (Srikantan etal., 2000). Further, the highly prostate tissue-specific expres-sion of PCGEM1 underscored its specific function in prostatebiology and physiology as well as its potential role in cancerbiology (Srikantan et al., 2000). The unique sequence charac-teristics of the PCGEM1 as a nonprotein coding RNA has pre-sented a conceptual challenge in studying its functions. The in-formation of genomes of humans and other organisms suggestthat an emerging number of such genes may have biologicalfunctions in diverse cellular processes (Eddy, 2001; Kapranovet al., 2002; Szymanski and Barciszewski, 2002; Morey andAvner, 2004; Shabalina and Spiridonov, 2004; Huttenhofer etal., 2005). It was recommended that ncRNAs can be dividedinto two classes: housekeeping RNAs, and regulatory RNAs(Morey and Avner, 2004). Housekeeping ncRNAs include ri-bosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nu-clear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs)(Morey and Avner, 2004). Regulatory ncRNAs include the mi-croRNAs (miRNA), which are usually 21 nucleotides in length,and large regulatory ncRNAs. According to this classification,PCGEM1 belongs to large regulatory ncRNAs. Structural andfunctional analyses of these large regulatory RNAs suggest po-tential roles in gene expression silencing, in stress response, andin tumorigenesis. H19, the best known example of a noncod-ing poly A� RNA involved in tumor biology, is expressed athigh levels in placenta and in tumors of embryonic tissues. H19RNA exhibited tumor suppressor activity when overexpressedin embryonic carcinoma cells (Ayesh et al., 2002). XIST andTSIX RNA molecules play critical role in X chromosome in-activation (Marahrens et al., 1998; Lee, 2000). BRCA1, a breastand ovarian tumor suppressor and nuclear protein, physically

associates with XIST RNA and influences the localization ofXIST on X chromosome (Ganesan et al., 2002, 2004). His-1and Bic represent examples of ncRNA implicated in the patho-genesis of hematological malignancies induced by oncogenicretroviruses (Askew et al., 1994; Tam et al., 1997), MALAT-1,a ncRNA with more than 8000 nucleotides in length, is signif-icantly associated with metastasis in early-stage nonsmall-celllung cancer (NSCLC) patients (Ji et al., 2003). NcRNA Tau-

REGULATION OF APOPTOSIS BY PCGEM1 139

FIG. 5. Effect of PCGEM1 overexpression on apoptosis intransiently transfected cells. (A) Northern blot analysis ofPCGEM1 overexpression in LNCaP cells after transfections (72 h) of pEAK8 and pEAK8-PCGEM1 vectors, respectively.(B–D) The PARP cleavage was analyzed in LNCaP cells (B),C4-2 cells (C), and C4-2b cells (D) transfected with controlvector pEAK8 (without PCGEM1 overexpression), and withPCGEM1 expression vector pEAK8-PCGEM1 (with PCGEM1overexpression) treated with/without DOX (0.2 �g/ml) for 24 h as indicated. The expression of tubulin served as an in-ternal control. (E) Quantification of relative band intensity ofcleaved PARP was normalized to the intensity of tubulin bandsusing Quantity One (version 4.5.0) computer program. Resultsare cumulative of three independent experiments. Asterisks in-dicate significant difference (p�0.05) from empty vector trans-fection.

rine Upregulated Gene 1 (TUG1) is necessary for the properformation of photoreceptors in the developing rodent retina(Young et al., 2005). Gene expression patterns and functionsof these ncRNAs are providing important insights into RNAbased mechanisms of gene expression, genomic imprinting, celldifferentiation, stress response, and tumorigenesis.

Our previous study demonstrated that PCGEM1 overex-pression in LNCaP as well as in NIH3T3 cells promotes cellproliferation and a dramatic increase in colony formation(Petrovics et al., 2004), suggesting a biological role ofPCGEM1 in cell growth regulation. The effect of PCGEM1overexpression on cell cycle was further analyzed using a panelof phosphorylation-specific antibodies raised against key cellcycle–related proteins. A significant increase in retinoblastomatumor suppressor protein (RB) phosphorylation (Ser807/811)was detected, indicating that PCGEM1 overexpression may af-fect cell proliferation through RB phosphorylation. This studyfurther demonstrated that p21Waf1/Cip1, an inhibitor of CDKs(Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993), wasdownregulated by PCGEM1 overexpression in LNCaP cells.Therefore, the decreased expression of p21Waf1/Cip1 in LNCaP-PCGEM1 cells might contribute to the increased phosphoryla-tion of RB through inhibiting CDK complexes.

p21Waf1/Cip1 levels are modulated by p53 and non-p53 path-ways (Johnson et al., 1994; Macleod et al., 1995; Yoshida etal., 1996). The next question we addressed was whetherPCGEM1 overexpression affects the induction of p21Waf1/Cip1

by p53. Our results demonstrated that the protein levels of bothp53 and p21Waf1/Cip1 were lower after 4-h DOX treatment ofLNCaP-PCGEM1 cells compared to control LNCaP cells. Thisimplies that the stabilization of p53 is at least in part inhibitedby PCGEM1 overexpression, which may lead to the down reg-ulation of p21Waf1/Cip1 in LNCaP-PCGEM1 cells.

Since p53 induces cell growth arrest as well apoptosis, theattenuated induction of p53 as well as p21Waf1/Cip1 lead us toexplore modulation of apoptosis by PCGEM1. Several lines ofexperimental evidence, including the modulation of cleavedcaspase 7 and cleaved PARP levels, as well as the percentageof apoptotic cells, demonstrated that the induction of apoptosisby chemotherapy agent DOX is inhibited by PCGEM1 over-expression in LNCaP cells. The inhibition of PARP cleavageby PCGEM1 overexpression was also observed in LNCaP cellstreated with three other chemotherapy agents: staurosporine,sodium selenite, and etoposide. The result suggests thatPCGEM1 may inhibit the functions of p53-dependent apoptoticmachinery.

Our previous report demonstrated that PCGEM1 expressionin LNCaP cells was regulated by androgen (Srikantan et al.,2000). PCGEM1 effects observed in LNCaP cells, an andro-gen-dependent prostate cancer cell line, might be different inandrogen independent prostate cancer cells. Our data indicatedthat PCGEM1 effects on apoptosis were not observed in twoandrogen-independent prostate cancer cell lines, C4-2 and C4-2b. The lack of PCGEM1 effects on apoptosis in these celllines might result from the feature of androgen independenceof these cells. Further evaluations are warranted to address thisissue.

In summary, we have unraveled a novel biologic function ofPCGEM1, a prostate-specific and cancer-associated ncRNAgene. By using stable and transient transfection of the widely

used LNCaP prostate cancer cell models, we demonstrate thatPCGEM1 inhibits apoptosis in LNCaP cells, in addition to itscell growth-promoting effects (Petrovics et al., 2004). HowPCGEM1 brings these functions remains to be determined. Thedata presented here and in previous studies highlight an in-triguing linkage between cell biologic functions of PCGEM1to cell growth and apoptosis, and provide novel insight intofunctions of a prostate tissue specific nonprotein-coding gene,whose expression is altered in prostate cancer cells. These func-tional readouts will also help us assessing structure–functionrelationships of PCGEM1. Our novel observations warrant fur-ther studies aimed at defining the mechanisms of PCGEM1functions in prostate cell biology and prostate cancer.

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Address reprint requests to:Shiv Srivastava, Ph.D., or Gyorgy Petrovics, Ph.D.

Department of SurgeryCenter for Prostate Disease Research

Uniformed Services University of the Health Sciences1530 East Jefferson Street

Rockville, MD 20852

E-mail: ssrivastava@cpdr.org or gpetrovics@cpdr.org

Received for publication October 3, 2005; received in revisedform October 26, 2005; accepted December 7, 2005.

REGULATION OF APOPTOSIS BY PCGEM1 141

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