Mutations of lysophosphatidic acid receptor-1 gene during progression of lung tumors in rats Takanori Yamada a , Yumi Obo a , Mami Furukawa a , Mayuko Hotta a , Ayako Yamasaki a , Kanya Honoki b , Nobuyuki Fukushima c , Toshifumi Tsujiuchi a, * a Laboratory of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502, Japan b Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan c Laboratory of Molecular Neurobiology, Department of Life Science, Faculty of Science and Technology, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502, Japan article info Article history: Received 10 November 2008 Available online 21 November 2008 Keywords: Lysophosphatidic acid receptor-1 Mutation Lung tumor Nitrosamine Rat abstract Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. In this study, mutations of lysophosphatidic acid receptor-1 (LPA1) gene were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitros- obis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNAs were extracted from paraffin-embedded tissues and exons 2–4 were exam- ined for mutations, using polymerase chain reaction (PCR)–single strand conformation polymorphism (SSCP) analysis. No LPA1 mutations were detected in 15 hyperplasias, but 2 out of 12 adenomas (16.7%) and 7 out of 17 adenocarcinomas (41.2%). These results suggest that mutations of LPA1 gene may be involved in the acquisition of growth advantage from adenomas to adenocarcinomas in lung car- cinogenesis induced in rats by BHP. Ó 2008 Elsevier Inc. All rights reserved. Lung cancer is one of the most common human malignancies, but the rate-limiting molecular events involved in its development remain largely unknown. The experimental model used in this study features the development of non-small cell lung cancers (NSCLCs) in rats given BHP in their drinking water, with high yields of adenomatous lesions, including adenocarcinomas [1,2]. As the step by step development of lung malignancies is accessible with this model, the molecular mechanisms involved can be readily investigated. Taking advantage of this model, we have been able to accumulate data on genetic and epigenetic alterations during carcinogenesis, including Ki-ras mutations [3], alterations in genes associated with transforming growth factor-b signaling pathway [4–6], and alterations in tumor suppressor genes located on human chromosome 3p [7–9]. Lysophosphatidic acid (LPA) is a bioactive mediator that induces diverse cellular effects, including regulation of cell proliferation, differentiation, transcellular migration, morphogenesis, and pro- tection from apoptosis [10–15]. LPA can induce cell proliferation, migration, invasion, and production of angiogenic factors in human ovarian cancer cell lines, suggesting that LPA may play an impor- tant role in the development of tumor cells [11,12,15–18]. LPA interacts with at least five G protein-coupled transmembrane receptors, lysophosphatidic acid receptor-1 (LPA1), LPA2, LPA3, LPA4, and LPA5 [17–20]. LPA1 is ubiquitously expressed in normal tissues, but the expressions of other LPA receptor subtypes are rel- atively restricted, suggesting these receptors may have different biological functions regarding LPA [10,17,18]. Recently, aberrant expressions of LPA1 have been reported in human and rat tumors, demonstrating that alteration of LPA1 expression might be impor- tant in the malignant transformation of tumor cells as well as LPA per se [11,12,15,16,21,22]. Moreover, we have reported that loss of LPA1 expression is due to its aberrant DNA methylation in rat tu- mor cell lines [23]. However, there have been few reports of LPA1 gene mutations in cancer cells. In the present study, we investigated LPA1 gene mutations in lung lesions, hyperplasias, adenomas, and adenocarcinomas in- duced in rats by BHP and evaluated the involvement of LPA1 alter- ations during rat lung carcinogenesis. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.044 Abbreviations: LPA1, lysophosphatidic acid receptor-1; BHP, N-nitrosobis(2- hydroxypropyl)amine; NSCLC, non-small cell lung cancer; PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism. * Corresponding author. Fax: +81 6 6723 2721. E-mail address: ttujiuch@life.kindai.ac.jp (T. Tsujiuchi). Biochemical and Biophysical Research Communications 378 (2009) 424–427 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc