[CANCER RESEARCH 61, 3741–3749, May 1, 2001] Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN Mieko Matsushima-Nishiu, Motoko Unoki, Kenji Ono, Tatsuhiko Tsunoda, Takeo Minaguchi, Hiroyuki Kuramoto, Masato Nishida, Toyomi Satoh, Toshihiro Tanaka, and Yusuke Nakamura1 Laboratories of Molecular Medicine [M. M-N., M. U., K. O., T. M., T. Ta., Y. N.] and Genome Database [T. Ts.], Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; Department of Obstetrics and Gynecology, School of Medicine, Kitasato University, Sagamihara 228-8555, Japan [H. K.]; Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba 305-8576, Japan [M. N.]; and Department of Obstetrics and Gynecology, Ibaraki Seinan Central Hospital, Tsukuba 306-0433, Japan [T. S.] ABSTRACT Akt/protein kinase B, cell survival, and cell proliferation (8). Over- expression of PTEN can decrease cell proliferation and tumorigenicity The PTEN tumor suppressor gene encodes a multifunctional phospha- (9, 10), an observation attributed to the ability of PTEN to induce cell tase that plays an important role in inhibiting the phosphatidylinositol-3- cycle arrest and apoptosis (11, 12). kinase pathway and downstream functions that include activation of Akt/protein kinase B, cell survival, and cell proliferation. Enforced ex- Thus, lack of PTEN expression may affect a complex set of pression of PTEN in various cancer cell lines decreases cell proliferation transcriptional targets. However, no systematic assessment of PTEN- through arrest of the cell cycle, accompanied in some cases by induction regulated targets in cancer cells has been reported to date. We used a of apoptosis. We used cDNA microarrays containing 4009 cDNAs to cDNA microarray to investigate transcriptional changes related to examine changes in gene-expression profiles when exogenous PTEN was PTEN signaling or secondary changes caused by the initiation of induced in PTEN-defective cells. The microarrays and subsequent semi- apoptosis or cell cycle arrest by transfecting wild-type PTEN into quantitative reverse transcription-PCR analysis revealed transcriptional cancer cells that were deficient in endogenous PTEN. Microarray stimulation of 99 genes and repression of 72 genes. Some of the differen- technology, which can permit analysis of the expression of thousands tially expressed genes already had been implicated in cell proliferation, of genes simultaneously, is a proven method for investigating gene- differentiation, apoptosis, or cell cycle control, e.g., overexpression of expression profiles in human cancers (13, 14). PTEN-induced transactivation of cyclin-dependent inhibitor 1B (p27Kip1) and 2B (p15INK4B), members of the TNF receptor family, tumor necrosis factor-associated genes, and members of the Notch-signaling and Mad families. To our knowledge this is the first report of transactivation of MATERIALS AND METHODS those genes by PTEN. The genes differentially expressed in our experi- Cell Culture. The 14 endometrial cancer cell lines used in this study are ments also included many whose correlation with cancer development had described in Table 1. Ishikawa3-H-12 (15, 16) was obtained from Tsukuba not been recognized before. Our data should contribute to a greater University (Tsukuba, Japan). HEC-1 (17), HEC-6 (18), HEC-50 (19), HEC59 understanding of the broad spectrum of ways in which PTEN affects (20), HEC-88 (20), HEC-108 (20), HEC-116 (20), and HEC-151 (21) were intracellular signaling pathways. Analysis of expression profiles with mi- obtained from Kitasato University (Sagamihara, Japan). AN3CA and KLE croarrays appears to be a powerful approach for identifying anticancer were obtained from the American Type Culture Collection (Manassas, VA), genes and/or disease-specific targets for cancer therapy. and Sawano (22), HHUA, and HOUA-I were obtained from the RIKEN Gene Bank (Tsukuba, Japan). Ishikawa3-H-12, Sawano, HEC-1, HEC-6, HEC-50, HEC59, HEC-88, HEC-108, HEC-116, HEC-151, and AN3CA cells were INTRODUCTION maintained in Eagle’s MEM with 10% FBS. HHUA and HOUA-I cells were The PTEN/MMAC12 gene on chromosome 10q23.3 has been iden- grown in Ham’s F12 medium with 10% FBS. KLE cells were grown in 1:1 tified as a tumor suppressor (1, 2). Deletions and mutations in PTEN mixture of DMEM and Ham’s F12 medium with 10% FBS. DNA Extraction and Mutation Screening. Genomic DNA was extracted occur frequently in advanced carcinomas of endometrium, breast, from each cell line using standard methods. All nine coding exons of PTEN bladder, and endometrioid ovarian tissue and also in glioblastomas were amplified from these DNA samples, and their nucleotide sequences were and malignant melanomas (1–4). Germ-line mutations of PTEN are determined by direct sequencing using BigDye Terminator RR Mix (Applied the cause of Cowden’s and Bannayan-Zonana syndromes of cancer Biosystems, Foster City, CA) on an ABI model 3700 DNA sequencer (Applied predisposition (5, 6). Biosystems) to reveal any mutations in the PTEN gene. A recent study demonstrated that PTEN acts as a phospholipid Construction of Recombinant Adenovirus. To construct AdCAPTEN phosphatase dephosphorylating the PtdIns (3,4,5)P3 and PtdIns viral vectors, the cDNA of PTEN was cloned by RT-PCR using placental (3,4)P2, resulting in PtdIns (4,5)P2 and PtdIns (4)P, respectively (7). mRNA as a template and primers 5Ј-CTT CAGCCACAGGCTCCCA-3Ј and It modulates the PI3K pathway by catalyzing degradation of the 5Ј-TGGTGTTTTATCCCTCTTGATA-3Ј. A 1.2-kb fragment of PTEN cDNA PtdIns (3,4,5)P3 generated by PI3K. This mechanism inhibits down- was cloned into the SwaI site of cosmid pAxCAwt (TaKaRa; Ref. 23), which contains the CAG promoter (composed of the cytomegalovirus enhancer and stream functions mediated by the PI3K pathway, such as activation of chicken ␤-actin promoter; Ref. 24) and an entire genome of type 5 adenovirus except for the E1 and E3 regions. Recombinant adenoviruses were constructed Received 4/27/00; accepted 2/28/01. by in vitro homologous recombination in the human embryonic kidney (HEK) The costs of publication of this article were defrayed in part by the payment of page cell line 293, using pAxCAPTEN and the adenovirus DNA terminal-protein charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. complex (TaKaRa; Ref. 23). As a control, AdCA viruses were generated from 1 To whom requests for reprints should be addressed, at Laboratory of Molecular cosmid pAxCAwt without a transgene. AdCALacZ viruses encoding the ␤-gal Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, gene under the control of the CAG promoter were constructed from the control 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: cosmid pAxCALacZ (TaKaRa). Viruses were propagated in the HEK293 cell 81-3-5449-5433; E-mail: [email protected]. 2 The abbreviations used are: PTEN, phosphatase and tensin homologue deleted on line and purified by two rounds of CsCl density centrifugation; viral titers were chromosome 10; MMAC1, mutated in multiple advanced cancers 1; PtdIns, phosphati- measured in a limiting-dilution bioassay using the HEK293 cells. In this study, dylinositol; P3, triphosphate; P2, biphosphate; PI3K, phosphatidylinositol-3-kinase; FBS, we used recombinant adenovirus stocks of fewer than five passages to mini- ␤ ␤ fetal bovine serum; MOI, multiplicity of infection; -gal, -galactosidase; MTT, 3, mize the possibility of replication-competent adenovirus contamination. (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal de- ␤ oxynucleotidyl transferase-mediated nick end labeling; RT-PCR, reverse transcription- -Galactosidase Transduction Assay. To assess efficiency of adenovirus- PCR; TNF, tumor necrosis factor; PI, propidium iodide. mediated gene transfer, 1 ϫ 105 cells from each culture were plated in 3741 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. GENE EXPRESSION PROFILES IN PTEN-TRANSFECTED CELLS Table 1 Characteristics of endometrial cancer cell lines (PTEN status and transduction efficiency) PTEN statusa Transduction efficiency (%)b Cell line Codon Mutation Predicted effect MOI 20 MOI 100 Sourcec HHUA 164 1bp(A) del Frameshift 48.2 89.7 1 289 1bp(A) del Frameshift HOUA-1 267 1bp(A) ins Frameshift 44.4 94.8 1 289 1bp(A) ins Frameshift Ishikawa 3-H-12 289 1bp(A) del Frameshift 66.5 100.0 2 317–318 4bp(ACTT) del Frameshift Sawano 16 2bp(AT) del Frameshift 27 94.8 2 130 CGA to GGA Arg(R) to Gly(G) HEC-1 None 79.3 100.0 3 HEC-6 intron 4(ϩ2) T to C Splice donor 44 79.5 3 289 1bp(A) del Frameshift HEC-50 None 43.5 100 3 HEC-59 41 TAC to CAC Tyr(Y) to His(H) 68.3 100.0 3 233 CGA to TGA Stop 246 CCG to CTG Pro(P) to Leu(L) 267 1bp(A) del Frameshift HEC-88 130 CGA to GGA Arg(R) to Gly(G) 95.5 100.0 3 173 CGC to TGC Arg(R) to Cys(C) 310 GAT to TAT Asp(D) to Tyr(Y) 341 TTT to TGT Phe(F) to Cys(C) HEC-108 6 2bp(AA) del Frameshift 66.7 100.0 3 289 1bp(A) del Frameshift HEC-116 Intron 2(Ϫ1) G to A Splice acceptor 57.6 100.0 3 173 CGC to TGC Arg(R) to Cys(C) 233 CGA to TGA Stop HEC-151 33 3bp(ATT) del In-frame deletion 78 100.0 3 76 2bp(AT) del Frameshift AN3CA 130 1bp(G) del Frameshift 0.9 5.3 4 KLE None 66.3 97.3 4 a PTEN mutations were detected by DNA sequencing. b Percentage of ␤-gal-positive cells 24 h after infection with AdCALacZ.