Molecular Cancer Therapeutics 361 Suppression of RAD21 gene expression decreases cell growth and enhances cytotoxicity of etoposide and bleomycin in human breast cancer cells Josephine M. Atienza,1 Richard B. Roth,1 Introduction 1 1 Caridad Rosette, Kevin J. Smylie, The RAD21 gene codes for a human homologue of Stefan Kammerer,1 Joachim Rehbock,2 1 1 Saccharomyces pombe Rad21 protein. The current knowledge Jonas Ekblom, and Mikhail F. Denissenko about this protein points to a role in modulation of cell 1Sequenom, Inc., San Diego, California and 2Frauena¨rzte growth and in cell defense against DNA damage, both Rosenstrasse, Munich, Germany processes being central to carcinogenesis. Several DNA repair genes including rad21 were initially identified in the fission yeast S. pombe as radiation-sensitive mutants (1, 2). Abstract Specifically, Rad21 has been implicated in homologous A genome-wide case-control association study done in our recombination–mediated double-strand break (DSB) re- laboratory has identified a single nucleotide polymorphism pair, and is unique among the radiation response genes in located in RAD21 as being significantly associated with that it also plays a role in cell cycle regulation (3, 4). Yeast breast cancer susceptibility. RAD21 is believed to function Rad21 and its mammalian homologue were subsequently in sister chromatid alignment as part of the cohesin complex identified as components of a conserved cohesin complex and also in double-strand break (DSB) repair. Following our (5, 6), which is believed to function in aligning sister initial finding, expression studies revealed a 1.25- to 2.5- chromatids during the early stages of cellular division. fold increased expression of this gene in several human Deletion of RAD21/Scc1/Mcd1 in mammalian cells leads breast cancer cell lines as compared with normal breast to abnormal separation of sister chromatids during tissue. To determine whether suppression of RAD21 interphase and improper alignment during metaphase. expression influences cellular proliferation, RNA interfer- These cells also incur increased levels of spontaneous ence technology was used in breast cancer cell lines MCF-7 chromosomal breaks and ionizing radiation–induced chro- and T-47D. Proliferation of cells treated with RAD21- mosomal aberrations, probably due to a reduction in DSB specific small inhibitory RNA (siRNA) was significantly repair efficiency (7). These observations suggest that reduced as compared with mock-transfected cells and cells RAD21 and the cohesin complex not only mediate the transfected with a control siRNA (Lamin A/C). This alignment of chromosomes in preparation for segregation inhibition of proliferation correlated with a significant into daughter cells during mitosis but also facilitate the reduction in the expression of RAD21 mRNA and with an repair of DNA damage incurred during DNA replication increased level of apoptosis. Moreover, MCF-7 cell sensi- by holding sister chromatids together. tivity to two DNA-damaging chemotherapeutic agents, DNA damage is a consequence of exposure to both etoposide and bleomycin, was increased after inhibition of exogenous and endogenous agents, resulting in a diverse RAD21 expression with a dose reduction factor 50 (DRF50) array of DNA modifications. Exogenous agents such as of 1.42 and 3.71, respectively. At the highest concen- ionizing radiation and radiomimetic chemicals and endog- trations of etoposide and bleomycin administered, cells enous agents such as oxygen radicals can all induce DSBs. transfected with a single siRNA duplex targeted against In addition, certain cellular processes including replication, RAD21 showed 57% and 60% survival as compared with meiosis, and V(D)J recombination also give rise to DSBs. control cells, respectively. Based on these findings, we DNA DSBs are among the most deleterious of DNA conclude that RAD21 is a novel target for developing cancer modifications, often resulting in mutagenesis or cytotox- therapeutics that can potentially enhance the antitumor icity (8). DSBs can contribute to tumorigenesis through the activity of chemotherapeutic agents acting via induction of introduction of mutations or chromosomal aberrations DNA damage. [Mol Cancer Ther 2005;4(3):361–8] leading to abnormal regulation of oncogenes or loss of tumor suppressor genes (9). Defects in DSB repair and increased levels of DSBs have both been linked to the development of human tumors (9). Several intricate and Received 9/10/04; revised 12/21/04; accepted 1/5/05. well-regulated DSB repair mechanisms have evolved to The costs of publication of this article were defrayed in part by the counteract these damages. DSBs can be repaired by payment of page charges. This article must therefore be hereby marked homologous recombination or by nonhomologous end advertisement in accordance with 18 U.S.C. Section 1734 solely to joining. indicate this fact. Many common anticancer agents introduce DSBs in Requests for reprints: Mikhail F. Denissenko, Sequenom, Inc., 3595 John Hopkins Court, San Diego, CA 92121. Phone: 858-202-9000; DNA. This therapeutic option takes advantage of the Fax: 858-202-9001. E-mail: [email protected] inefficient DNA-damage checkpoints in cancerous cells Copyright C 2005 American Association for Cancer Research. and, therefore, their compromised ability to efficiently Mol Cancer Ther 2005;4(3). March 2005 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. 362 RAD21 Suppression Inhibits Growth of Breast Cancer Cells repair the damage caused by the drugs before cell division, competitor oligonucleotide that differs at one base position resulting in increased cytotoxicity. Disabling the cellular from the cDNA target. This competitor is used to calibrate processing and repair of DSBs potentiates cell sensitivity to the assay and quantitate the genes of interest at an absolute traditional chemotherapeutic agents (10–12). Because hu- level by matrix-assisted laser desorption-ionization time-of- man RAD21 is believed to play a dual role in the cell, both flight mass spectrometry (MALDI-TOF) mass spectro- in modulation of sister chromatid alignment during cell metry. The principle of Quantitative Gene Expression by cycle and through repair of DNA DSBs, it presents a good MassARRAY assay was described elsewhere (14). Levels of target candidate for adjuvant gene-specific approaches that gene-specific mRNA were normalized against levels of 18S enhance sensitivity of tumor cells to anticancer agents. rRNA by dividing the observed RAD21 transcript concen- In a large-scale breast cancer case-control study, we tration by the observed concentration of 18S rRNA for each obtained evidence that RAD21 is a susceptibility gene for respective sample. The following oligonucleotides were breast cancer (13). In this article we investigated the role of used: 5V-ACGTTGGATGATATGGATGAGGATGA- RAD21 in cancer cell proliferation by analyzing its TAATGTATC-3V (forward PCR primer), 5V-ACGTTG- expression in a number of human breast tumor cell lines GATGCAGTCATGGTTGGCATTG-3V (reverse PCR and also exploring the utility of silencing its expression in primer), 5V-GTTCAACGGGATCCACTGAAT-3V (MassEX- these cells. Here we present the first report showing that TEND primer), 5V-CAGTCATGGTTGGCATTGGTT- inhibition of RAD21 gene expression in breast cancer cells CAACGGGATCCACTGAATCAGGACTATCAGGCC- results in decreased cellular proliferation, increased apo- CACCCATTGATACATTATCATCCTCATCCATAT-3V ptosis, and increased cell killing after exposure to chemo- (cDNA competitor). Analysis included triplicate experi- therapeutic agents. ments with quadruplicate spotting of reaction products onto MALDI-TOF chips. Data shown represent means of three experimental measurements. Materials and Methods Small InhibitoryRNA Design Cell Lines and Culture Conditions siRNA duplexes were designed according to the guide- MCF-7, T-47D, Hs 578Bst, ZR-75-1, HCC1937, Au-565, Hs lines of Elbashir et al. (15). The following siRNA against 578, BT-474, HCC1395, HCC1428, HCC1500, and SK-BR-3 RAD21 were used: siRad21_272, 5V-AAGCCCAUGU- were acquired from American Type Culture Collection GUUCGAGUGUA-3V; siRad21_1175 5V-AAGAGUUG- (Manassas, VA). Human mammary epithelial cells were GAUAGCAAGACAA-3V, and scrambled siRad21_1175 generously provided by S. Bates (City of Hope Medical siRNA, siRad21_1175s 5V-AAGACAGAUACGAUGAU- Center, Duarte, CA). MCF-7 and T-47D were maintained GAGA-3V. The sequence of Lamin A/C control siRNA in MEME (American Type Culture Collection) and RPMI duplex was 5V-CUGGACUUCCAGAAGAACA-3V. Ready to (American Type Culture Collection) media, respectively, use synthetic siRNA duplexes, including a fluorescent supplemented with 10% fetal bovine serum (Omega transfection control Cy3-modified Luciferase (GL2) siRNA, Scientific, Tarzana, CA) and 10 Ag/mL insulin (Invitrogen, were purchased from Dharmacon (Lafayette, CO) and, Carlsbad, CA). Cells were fed every 5 days and subcultured upon dilution in water, were directly used in transfection when confluent. experiments. RNA, Reverse Transcription ^ PCR, and Quantitative Cell Proliferation and Apoptosis Assays Gene Expression Analysis MCF-7 and T-47D cells were plated in six-well plates Human normal breast tissue total RNA was purchased at 3.5 Â 105 and 2.5 Â 105 cell/mL concentrations, from Ambion (Austin, TX) and used for cDNA synthesis. respectively. Twenty-four hours after plating (day 0), cells To assess cellular expression of RAD21 mRNA, 50 Â 106 were transfected