BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 154 – 160 (1997) ARTICLE NO. RC976310 Overexpression of ZBP-89, a Zinc Finger DNA Binding Protein, in Gastric Cancer Toshifumi Taniuchi,* Eric R. Mortensen,* Amy Ferguson,† Joel Greenson,† and Juanita L. Merchant* , ‡ ,1 *Department of Internal Medicine and Physiology, and †Department of Pathology, University of Michigan, and ‡Howard Hughes Medical Institute, Ann Arbor, Michigan 48109 Received February 11, 1997 tion factor Sp1 occurs in some gastric cancers, appears ZBP-89 is a Kru ¨ ppel-type zinc finger protein that to correlate with EGF receptor overexpression and may binds to the gastrin EGF response element (gERE). Sp1 be a marker for transformation (3). Sp1 is also ex- binds to the same DNA element and transactivates gas- pressed in the developing mouse stomach (4) and binds trin promoter activity, whereas ZBP-89 competes for to GC-rich elements present in the promoters of several Sp1 binding and prevents EGF induction. Both tran- growth-related genes including TGFa, the EGF recep- scription factors mediate growth factor signals origi- tor, TGFb and c-erbB2 (5-9). Thus Sp1-mediated gene nating from the EGF receptor and thus were studied expression is potentially one mechanism by which can- in normal and neoplastic tissues or cell lines. When cers regulate the overexpression of growth factor recep- compared to normal tissue from the same patient, ZBP- tors and ligands. 89 protein expression was increased in neoplastic tis- Recently, we have found that Sp1 and a second zinc sue from the stomach antrum and in malignant cell finger protein called ZBP-89 bind to a GC-rich EGF lines. RT-PCR analysis of ZBP-89 mRNA correlated response element in the human gastrin promoter (10). with protein overexpression. Immunocytochemical Gastrin is a hormone expressed in the stomach antrum studies confirmed that ZBP-89 expression is elevated that stimulates gastric acid secretion and epithelial cell in neoplastic tissue and chronic gastritis, whereas Sp1 expression was nearly unchanged. These results sug- growth (11). Changes in gastric pH, mucosal inflam- gest that the transcription factor ZBP-89, like Sp1, may mation and developmental cues stimulate and repress be a marker for neoplastic transformation in some gas- gastrin gene expression (12,13). Gastrin is expressed tric cancers.  1997 Academic Press by some pancreatic and colon cancers presumably as an autocrine growth factor for these malignancies (14- 16). Consistent with the positive and negative expres- sion of this hormone, Sp1 and ZBP-89 respectively Gastric cancer is the most common cancer in the stimulate and inhibit gastrin gene expression (10,17). world and is the leading cause of cancer deaths in de- Since ZBP-89 is a novel DNA binding protein that mod- veloping countries (1). Nevertheless, morphologic clas- ulates growth factor signals, we studied the expression sifications and molecular markers are not as precisely of this factor in cell lines and gastric malignancies us- defined as for colon cancer. Gastric cancers are classi- ing biochemical and immunocytochemical methods. fied as well-differentiated adenocarcinoma or poorly- differentiated adenocarcinoma and represent the accu- MATERIALS AND METHODS mulation of multiple genetic alterations (2). These al- terations vary depending upon the histologic subtype, Cell culture and tissue procurement. Normal human epidermal mel- indicating that these subtypes may evolve along differ- anocytes (NHEM), normal human bronchial epithelial cells (NHBM) ent genetic pathways. Overexpression of the transcrip- and normal human mammary epithelial cells (NMEC) were purchased from Clonetics (San Diego, CA) and cultured according to the suppliers instructions. All cell lines were purchased from American Tissue Cul- ture Center except the MKN-45 cell line which was a kind gift from 1 Corresponding author: Juanita L. Merchant, 3510 MSRB I, 1150 West Medical Center Drive, Ann Arbor, MI 48109. Fax: (313) 936- Dr. E. Tahara (18) and breast cancer cell lines Hs578t and SKBR3 which were gifts from Dr. Eric Fearon (University of Michigan Cancer 1400. E-mail: merchanj@umich.edu. Abbreviations used: RT-PCR, reverse transcriptase-polymerase Center). The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY) containing 8% horse chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 154