Allelic loss and mutations in a new ETRG-1 gene are early events in diethylstilbestrol-induced renal carcinogenesis in Syrian hamsters Kamaleshwar P. Singh a, ⁎ , Deodutta Roy b a Department of Biology, Texas Southern University, Houston, TX 77004, USA b Department of Environmental and Occupational Health, Florida International University, Miami, FL 33199, USA Received 26 June 2007; received in revised form 14 October 2007; accepted 15 October 2007 Received by M. Di Giulio Available online 26 October 2007 Abstract Diethylstilbestrol (DES) is a synthetic estrogen and well known human carcinogen, but the mechanism by which DES causes cancer is not clear. In this study, using AP-PCR method we have identified a genetic alteration that was common in DES-induced kidney tumors as well as in its surrounding non-tumor tissue. Further characterization of this genetically altered region revealed that it represents an uncharacterized novel gene. We have named this gene as Estrogen Target Rodent Gene-1 (ETRG-1). Southern blot analysis revealed about 50% reduction in the ETRG-1 gene-specific hybridization signal, indicating genomic loss of one allele of ETRG-1 in DES-induced tumors as compared to its age-matched control. Sequence analysis of the remaining allele of ETRG-1 from tumor revealed point mutations and an insertion of 37 bp as compared to its normal allele from the age-matched control kidney tissue. The expression of ETRG-1 at transcript level was found to be extremely reduced or undetectable in DES-induced kidney tumors as compared to its age-matched control kidney tissue. Thus, the findings of this study suggest that (a) DES-induced allelic loss and mutations may be involved in DES-induced kidney carcinogenesis, (b) Constitutive expression of ETRG-1 in normal kidney tissue and decreased or undetectable expression in tumors, presumably as a result of allelic loss and mutational inactivation, suggest that it may be a tumor suppressor gene, and finally, (c) The identification of the allelic loss and mutations that were common in DES-exposed non-tumor kidney tissue, and in frank kidney tumors indicate that these genetic aberrations are early events in DES-induced kidney carcinogenesis and it may serve as biomarker for early detection of DES-induced cancer. © 2007 Elsevier B.V. All rights reserved. Keywords: Mutation; Allelic loss; Estrogen-induced cancer; Novel gene; Diethysltilbestrol; RAPD 1. Introduction Stilbene estrogen (diethylstilbestrol, DES) is a well-known carcinogen in experimental animals as well as in human (IARC, 1999). Recent epidemiological studies suggest a slightly in- creased threat of breast cancer in women exposed to DES (Palmer et al., 2006). The treatment of male Syrian hamsters with natural estrogen, 17β-estradiol (E 2 ) or DES produces 90– 100% incidence of kidney tumors (Kirkman, 1972; Li and Nandi, 1990; Roy and Liehr, 1990).The very few target genes involved in pathologies linked to DES have been identified. For example, the early activation of Helicase-Like Transcription Factor (HLTF/SMARCA3) in DES-induced renal carcinogenesis has been reported (Debauve et al., 2006). Developmental ex- posure to DES results in altered expression of genes associated with cell growth, differentiation, and adhesion, such as, TGFβ, CyclinD1, SFRP4, in uterine of mice (Newbold et al., 2007). Deregulation of cell cycle components Cyclin D1, Cyclin E, and pRb in early and frank estrogen-induced renal neoplasias in the Available online at www.sciencedirect.com Gene 408 (2008) 18 – 26 www.elsevier.com/locate/gene Abbreviations: BLAST, basic local alignment search tool; cDNA, DNA complementary to RNA; DES, diethylstilbestrol; DNase, Deoxyribonuclease; dNTP, deoxyribonucleoside triphosphate; ETRG-1, Estrogen Target Rodent Gene-1; LOH, loss of heterozygosity; ORF, open reading frame; RACE, rapid amplification of cDNA ends; RAPD, random amplified polymorphic DNA; RNase, ribonuclease; RFLP, restriction fragment length polymorphism. ⁎ Corresponding author. Department of Biology, Texas Southern University, 3100 Cleburne St., Houston, TX 77004, USA. Tel.: +1 713 313 1028; fax: +1 713 313 7932. E-mail addresses: Kamaleshwar9@aol.com, Singhk@tsu.edu (K.P. Singh). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.10.022