The Expression of Truncated MK in Human Tumors Tadashi Kaname,* , ² ,1 Kenji Kadomatsu,² Kuniaki Aridome,‡ Shin-ichi Yamashita,§ Kiyoshi Sakamoto,§ Michio Ogawa,§ Takashi Muramatsu,² and Ken-ichi Yamamura* *Institute of Molecular Embryology and Genetics and §Department of Surgery, Kumamoto University School of Medicine, Kuhonji 4-24-1, Kumamoto 862, Japan; ²Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Shouwa-ku, Nagoya 466, Japan; and ‡Department of Surgery, Kagoshima University, Faculty of Medicine, Sakuragaoka 8-35-1, Kagoshima 890, Japan Received December 25, 1995 Midkine (MK) is a heparin binding growth/differentiation factor different from fibroblast growth factors (FGFs), and is largely composed of two domains which are formed by a folded polypeptide chain interconnected by disulfide bridges. Polymerase chain reaction revealed the presence of a short MK mRNA in 7 among 12 human tumor cells which expressed MK mRNA. All of 4 pancreatic carcinoma cell lines expressed the short species in addition to the full size mRNA. The short mRNA lacked an exon and resulted in the lack of a more N-terminally located domain. The truncated form of MK was found also in some surgically removed specimens of human tumors, but not in noncancerous tissue. © 1996 Academic Press, Inc. Midkine (MK) is a novel growth factor found as a product of a retinoic acid responsive gene, and has neurotrophic activities (1–4). It consists of 5 exons (5) and is located on chromosome 11q11.2 in human (6). MK mRNA is strongly expressed in many human carcinoma cells (7,8). In the case of Wilms’ tumor, all 6 of surgically removed specimen showed intense MK expression (7) and anti-MK antibody partly inhibited growth of cultured tumor cells (4), indicating that it plays a role in enhanced growth of Wilms’ tumor cells. Altered forms of growth factors or products of tumor suppresser genes are often produced in tumor cells by alternative splicing, and they play key roles in tumorigenesis (9). So far it is not known whether altered MK structure is present in tumor cells or normal tissues. This paper demonstrates altered processing of MK mRNA in human tumor cells. MATERIALS AND METHODS Tumor Cell Lines The three lung adenocarcinoma cell lines (A549, PC-3, RERF-LCOK), the gastric adenocarcinoma cell line (NUGC-3), the hepatoma cell line (HuH7), two lymphoma cell lines (U937, Molt4), and the Wilms’ tumor cell line (G401) were obtained from the Japanese Cancer Research Resource Bank (Tokyo, Japan). The pancreas adenocarcinoma cell lines (AsPC-1, BxPC-3, Capan-1) were obtained from the American Type Culture Collection (Rockville, MD). The esophagus carcinoma cell line (TEN-8) (ref. 10) and the pancreas carcinoma cell line (SUIT-2) (ref. 11) were generously offered from Department of Surgery, Tohoku University and Department of Surgery, Miyazaki Medical College respectively. The two cell lines GaCa (12) and GBK-1 were established from human gastric adenocarcinoma and gall bladder carcinoma respectively. RNA Isolation The human cell line G401 was cultured in McCoy 5A, 10% FCS. The other cell lines were cultured in RPMI 1640, 15% FCS, and were harvested by 0.25% trypsin-EDTA. Tumor specimens and the normal tissues were surgically removed from cancer patients. Total cellular RNA from all cell lines, tumors, and normal tissues was extracted using guanidine- thiocyanate-phenol-chloroform method (13). 1 Corresponding author. FAX.: 81-96-373-5321. e-mail: tkan@gpo.kumamoto-u.ac.jp. Abbreviations: MK, midkine; RT–PCR, reverse transcript–polymerase chain reaction; GAPDH, glyceraldehyde 3-phos- phate dehydrogenase. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 219, 256–260 (1996) ARTICLE NO. 0214 256 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.