[CANCER RESEARCH 50. 6146-6153. October I, 1990] Two Chromosomal Locations for Human Ornithine Decarboxylase Gene Sequences and Elevated Expression in Colorectal Neoplasia1 Diane M. Radford,2 Hiroshi Nakai,3 Roger L. Eddy, Linda L. Haley, Mary G. Byers, W. Michael Henry, David D. Lawrence, Carl W. Porter, and Thomas B. Shows4 Departments of Surgical Oncology [D. M. R.]. Human Genetics [R. L. E., L. L. H., M. G. B., W. M. H., T. B. S.J, Experimental Therapeutics [C. W. P.], and Biomathematics [D. D. L.], Koswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263 ABSTRACT et al. (14) reported elevated polyamine levels in colon cancers independent of site, stage, and degree of differentiation of the The polyamines are known to be essential for cellular proliferation. tumor. Bile salts, thought to be tumor promoters in the human Ornithine decarboxylase (OIK) is a rate-limiting enzyme in the synthesis colon, induce ODC activity in rat colon mucosa (15). The of these amines, and activity is elevated in colorectal tumors and polyps. activity of S-adenosylmethionine decarboxylase, also a rate- Two ODC genes (designated OIK I and ODC2) were localized by somatic cell hybridization and in situ techniques to 2p25 and 7q31-qter, limiting enzyme in polyamine biosynthesis, is elevated in colon respectively. Investigation of the expression of ODC in colorectal neo neoplasia relative to adjacent mucosa (11). We have investi plasia reveals a consistent increase in niKNA expression compared with gated amplification and polymorphism of the 5-adenosylme- normal adjacent mucosa and control mucosa, ranging from 1.3- to 12.2- thionine decarboxylase gene in colonie neoplasia and have fold. No amplification of the loci was seen. Comparison of ODC mRNA localized the gene to sequences on chromosomes 6 and X (16). expression with ODC activity from the same samples revealed no direct ODC gene sequences have been assigned to chromosome correlation, suggesting that regulation of ODC in this system occurs at 2pter —»p23and 7cen —>7qter (17). In this study, we have the posttranscriptional level. localized the ODC loci to specific sites by somatic cell and in situ techniques. We have also investigated the expression of INTRODUCTION ODC mRNA in matched samples of colorectal carcinoma and uninvolved mucosa, and in benign polyps compared with mu The polyamines putrescine, spermidine, and spermine are cosa, to elucidate the nature of regulation of ODC in neoplasia. low-molecular-weight bases known to be essential for cellular proliferation and differentiation (1, 2). A rate-limiting step in MATERIALS AND METHODS the biosynthesis of the polyamines, namely the decarboxylation of ornithine to putrescine, is catalyzed by the enzyme ODC.5 Probes The importance of ODC in intestinal neoplasms has been ODC. The human ODC cDNA probe, pODC10/2H, was a gift from elucidated by a number of authors. Its activity is elevated in O. Janne (17). The cDNA probe is 1825 nucleotides long with an open tumors compared with normal tissue (3). Takano et al. (4) reading frame of 1383 nucleotides and was inserted into the EcoRl site found an early rise in ODC activity following the intrarectal ofpBR322(18). administration of a carcinogen to rats. The incidence of colon TPI. TP1 is a housekeeping enzyme active in glycolysis, gluconeo- tumors in rats (5) and mice (6) after intrarectal instillation of a genesis, and the pentose shunt. The cDNA probe for TPI, TPI-5A, was carcinogen is significantly reduced when DFMO, an irreversible a gift of L. Maquat (19, 20). This cDNA contains the last 2 nucleotides inhibitor of ODC (7), is also administered. of the translation initiation codon. the entire 744-nucleotide coding region, and the 448-nucleotide 3' untranslated region. Luk and Baylin (8), investigating ODC activity in patients with familial polyposis, reported a step-wise increase in ODC Somatic Cell Hybridization. The chromosomal location of the ODC gene was determined by human-mouse somatic cell hybrid techniques activity in benign colonie polyps as the degree of dysplasia (21, 22) using isolated cell hybrids as described (23, 24). Human increased. Colonie polyps are known to be premalignant lesions, chromosomes were identified in cell hybrids by chromosome analysis with the incidence of cancer increasing as the size of the polyp and mapped enzyme markers, and partly by mapped DNA probes (21, increases (9, 10). Porter et al. (11) also found an increase in 22, 25). DNA from these cell hybrids with different numbers and ODC activity in colonie carcinomas approximately 8-fold combinations of human chromosomes was isolated by methods de greater than in adjacent noninvolved mucosa, with the activity scribed by Naylor et al. (26). in benign polyps being intermediate between mucosa and car After isolation, DNA from 30 cell hybrids was digested with the cinoma. An increase of ODC activity in colorectal cancers and restriction endonuclease Pstl under the conditions prescribed by the polyps compared with normal tissue was also reported by manufacturer (Boehringer Mannheim). These hybrids were derived LaMuraglia et al. (12) and Moorehead et al. (13). Kingsnorth from 13 unrelated human and 4 mouse cell lines. The enzyme Hindlll was used to digest DNA from 35 cell hybrids involving 13 unrelated Received 7/21/89; accepted 6/26/90. human and 4 mouse cell lines. Digested DNA fragments were separated The costs of publication of this article were defrayed in part by the payment by electrophoresis in 0.8% agarose and transferred to Zetapor (AMF of page charges. This article must therefore be hereby marked advertisement in Cuno) by the Southern technique (27). Filters were baked for 3 h, then accordance with 18 U.S.C. Section 1734 solely to indicate this fact. washed in 0.1 x SSC, 0.5% SDS for l h at 65°C.The nick-translated ' This investigation was supported by NIH Grants CA 28853 and CM 20454, and American Cancer Society Grant CD62. ODC probe was hybridized to the filters for 48 h under conditions 1 Present address: Department of Surgery, St. Louis University Hospital, 3635 described by Prowse et al. (28). Filters were washed under low strin Vista at Grand. P. O. Box 15250, St. Louis, MO 63110. gency conditions (1x SSC, 0.1 % SDS) for l h at 60°C.Autoradiographs 3 Present address: Department of Pediatrics, Tohoku University, School of were obtained after exposure at -70°C for 3-5 days. The filters were Medicine. 1-1 Seiryo-machi, Sendai. 980 Japan. ' To whom requests for reprints should be addressed, at Department of Human then washed under high stringency conditions (0. Ix SSC, 0.1% SDS) Genetics, Roswell Park Memorial Institute, New York State Department of at 68°Cfor 1 h. After 7-14 days at —70°C,autoradiographs were Health, Buffalo, NY 14263. 5The abbreviations used are: ODC, ornithine decarboxylase; DFMO, difluo- developed. Filters containing only human DNA were rinsed at room temperature in 500 ml of 2x SSC and 0.1% SDS, then washed in 0.1 x romethylornithine; TP1, triosephosphate isomerase; cDNA, complementary SSC and 0.1% SDS at 50°C. DNA; SSC. standard saline citrate; SDS, sodium dodecyl sulfate. 6146 Downloaded from cancerres.aacrjournals.org on October 9, 2021. © 1990 American Association for Cancer Research. ODO GENE MAPPING, AND COLORECTAL NEOPLASIA In Situ Hybridization, ¡nsitu hybridization was performed using techniques described by Zabel et al. (29). Long prometaphase chromo 12345 MH somes were hybridized to 'H-labeled ODC cDNA probe by the method of Harper and Saunders (30). Silver grains produced from photographic emulsion revealed the presence of the hybridized probe. Chromosome -9.9kb spreads were then stained with Giemsa for G banding. Tissue Procurement. Tissue samples were obtained from surgical specimens at Roswell Park Memorial Institute. Tissue for analysis was cut by the pathologist soon after removal from the patient. Samples were taken of adenocarcinoma and/or benign polyps and adjacent noninvolved mucosa approximately 5 cm away from the lesion. Tissue was frozen in liquid nitrogen and stored at -70°C for later extraction 5.5 of DNA and RNA. Patients gave written consent for inclusion in the 4.6 study. The histológica! nature of the specimen received was confirmed by the pathologist. DNA Extraction. Samples of whole blood were obtained from vol unteers in the Red Cross. WBC were isolated from heparinized blood using the dextran sedimentation method (31). High-molecular-weight chromosomal DNA was extracted as described by Naylor et al. (26). -2.8 Tissue samples were thawed, homogenized, incubated with SDS and proteinase K, and extracted with phenol followed by chloroform/ asoamyl alcohol; 10 ng of each DNA sample were digested as described above. RNA Extraction. RNA was extracted by the guanidine isothiocyanate method as described by Tricoli et al. (32). Northern Blotting. Gene expression was investigated by Northern glyoxal gel analysis (33). Ten ng of RNA were loaded into 1.2% agarose gels, and electrophoresis was performed for 4-5 h at 90 V with circu ***« lating buffer. RNA was transferred to Zetabind (AM F Cuno) for hybridization to the cDNA ODC and TPI probes. Hybridization con ditions were as described by Tricoli et al. (32) and wash conditions were as described by Church and Gilbert (34). Scanning densitometry pro vided quantitation of the ODC and TPI signals on the autoradiographs. Statistical Analysis. Nonparametric statistics were calculated because the data distribution was not normal for tumor RNA, adjacent mucosa RNA or fold increases. Computations were made with Statistical Pack age for the Social Sciences. Comparisons of RNA levels between Dukes' Pstl HIGH STRINGENCY Fig. 1. Southern hybridization of cDNA probe pODC10/2H to Pst\ digests of stage, tumor primary site, and degree of differentiation were performed DNA from human (//).