[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
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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 (//). mouse (M), and somatic cell hybrids (Lanes 1-5). High with a Mann-Whitney test. Elevation of matched pairs of tumor and stringency wash conditions; lane 3 is positive for the 5.5-kilobase band; Lanes 1, adjacent normal mucosa was done with a Spearman rank-order corre 3, and 5 are positive for the 2.8-kilobase band; and Lanes I and 5 are very weakly lation and Wilcoxon matched-pairs test. positive for the 8.4-kilobase band and are often difficult to score in cell hybrids. Lengths of DNA bands are indicated in kilobase (right). ODC Activity. ODC activity was assayed as described by Porter et al. (11). Table 1, the 8.4- and/or 5.5-kb bands segregated together with the 2.8-kilobase band (only cell hybrids were included that could RESULTS unequivocally be scored for these 3 bands). These 3 bands were Gene Mapping distributed in cell hybrids that without exception retained hu man chromosome 2. All other human chromosomes segregated Somatic Cell Hybrids. The chromosomal assignment of ODC independently of the 3 bands. Therefore, sequences for these 3 was determined by correlating the presence or absence of spe bands were encoded on human chromosome 2. cific human chromosomes in a panel of human-mouse somatic Regarding the 9.9-kilobase band (Fig. 1), experiments using cell hybrids with ODC sequences in the DNA isolated from the human-mouse hybrid DUA-1CSAGF, which has retained these hybrid cell lines. The human ODC DNA probe (pODC 10/ only human chromosome 7, revealed that the 9.9-kilobase band 2H) hybridized to 6 Pstl fragments of human cell line DNA. on Pstl digests was present in this hybrid and therefore this These fragments measured 9.9, 8.4, 5.5, 4.6, 2.8, and 2.1 band mapped to human chromosome 7 (detailed mapping of kilobases long. Under conditions of high stringency, the 9.9-kb ODC sequences to chromosome 7 is described below). band and the 2.1-kb band (seen in a later illustration) became In Situ Hybridization. In situ hybridization was effective in less prominent (Fig. 1). In cell hybrids, these bands, in addition localizing the sequences to a specific site on chromosome 2. Of to the 8.4-kb and 4.6-kilobase bands, could not be scored easily the 198 metaphase chromosome sets examined, 361 attached due to either overlapping mouse bands or faintness of the signal. silver grains were seen with 58 grains (16.1%) touching chro In Fig. 1, lanes 1, 3, and 5 are positive for the 2.8-kilobase mosome 2. Thirty-one % of the grains on 2 were located at band; Lane 3 is positive for the 5.5-kb band; and Lanes 1 and 5 2p25 (Fig. 2). Fourteen grains were distributed on chromosome contain hybrids weakly positive for the 8.4-kilobase band. 7 but never above background levels at any site. No significant Analysis of 30 mouse-human cell lines and DNA from unrelated distribution of grains was observed on any human chromosome individuals demonstrated that the 8.4- and 5.5-kilobase bands except chromosome 2 at p25. were alÃelesat the same locus (below, and Fig. 3), and these 2 Chromosome 2 Restriction Fragment Length Polymorphism. bands were counted together for scoring purposes. As shown in Pstl digests of leukocyte DNA from 40 unrelated individuals 6147
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ODC, GENE MAPPING. AND COLORECTAL NEOPLASIA 1 2 3 Kb 9.9-m m m 8.4- 2p25 5.5- 4.6- 2B mm* 2.1- 3«2 24a Fig. 3. Banding pattern of DNA hybridization with pODC10/2H in a human sample after a /Vl digest. Only 3 patterns are seen in 40 samples examined. In all samples, the 9.9-kilobase band (chromosome 7) and the 4.6-, 2.8-, and 2.1- kilobase bands remain constant. In Lane 1, the 8.4-kilobase band is absent, representing the 5.5-kilobase homozygote. In Lane 2, the 5.5-kilobase band is absent, representing the 8.4-kilobase homozygote. In Lane 3 both bands are present, representing the 8.4-/5.5-kilobase hétérozygotephenotype. stringency, 2 fragments of human DNA hybridized to the ODC probe measuring 5.5 and 4.0 kb (Fig. 4). The 5.5-kilobase band was positive in Fig. 4, Lanes 1, 2, and 3, and the 4.0-kilobase band was positive in Fig. 3, Lane I. ODC Table 2 reveals that the 5.5-kilobase band was located on Fig. 2. /;/ situ hybridization with pODC10/2H. Distribution of silver grains chromosome 2, and the 4.0-kilobase band on chromosome 7. localizing ODC on chromosome 2 from 198 metaphases. There were 56 meta- The hybrid JSR-17S, with the 7/9 translocation [7pter -» phase cells with grains on chromosome 2 (29.3%); 31% (18 of 58) of the grains 7q22:9p23 —»9pter],was negative for this band, which localized on chromosome 2 are located at 2p25. A representative metaphase chromosome 2 is shown with labeling of 2p25 (arrow). There was no significant accumulation the 4.0-kilobase band to the q22 —»qterregion of 7. The hybrid of silver grains above background at any other chromosomal site, including VTL-7, also negative for the 4.0 band, had a deletion of 7 and chromosome 7. had retained only the 7pter—>7q31 region. The smallest deleted region would have indicated a further localization of the 4.0- revealed 3 consistent banding patterns initially suggested by the kilobase sequence to the q31 —»qterregion of chromosome 7. mapping data (Fig. 3). In all individuals, the 9.9-kilobase band (localizing to chromosome 7, see below) was present. The 2.8- DNA and RNA Analysis of Coloréela)Neoplasia kilobase band on chromosome 2 was also constant, as was the 2.1-kilobase band. The 4.6-kilobase band, which could not be DNA Analysis. In all, 18 colorectal carcinomas and 7 colo- used for scoring purposes, was seen very faintly. The 8.4- rectal polyps were examined. The tumor was well differentiated kilobase and 5.5-kilobase bands mapping to chromosome 2 in 1 patient, moderately differentiated in 12, and poorly differ showed a DNA restriction fragment length polymorphism (35) entiated in 5. Site was distributed as follows: rectum, 7 and since they segregated between individuals as alÃelesat the same sigmoid/descending colon, 4; transverse, 3 and cecum 4. Dukes' locus. These phenotypes constituted at least a 2-allele locus stage of the primary tumor (36) was Stage BI in 4 patients, with the 5.5-kilobase homozygote in Fig. 3, Lane /, the 8.4- Stage BII in 5, Stage CI in 6, and Stage CII in 3. Of the 7 kilobase homozygote in Fig. 3, Lane 2, and the 8.4-/5.5-kilobase benign polyps, 6 were \ ¡lionsadenomas with mild-to-moderate hétérozygoteinFig. 3, Lane 3. Of 40 individuals, 3 corre atypia, and 1 was recorded as a tubular adenoma with atypia. sponded to the 5.5-kb homozygote, Fig. 3, Lane 1; 21 corre Tumor or polyp DNA was run alongside matched pathologi sponded to the 8.4-kb homozygote, Fig. 3, Lane 2; and 16 cally normal adjacent mucosa DNA from the same patient and corresponded to the 8.4-/5.5-kilobase hétérozygote.Fig.3, from normal controls. No amplification of either ODC locus Lane 3. The alÃelefrequencies calculated by the Hardy Weinberg was seen in either carcinoma or polyp with either Pst\ or ///Will law were 5.5 (p) = 0.28 and 8.4 (q) = 0.72. digests after Southern blot analysis (27) (data not shown). Chromosome 7 ODC Segment. The DNA of 35 human-mouse In 1 patient, CCI 5, there was a difference between tumor and hybrids was digested with ///Will. Under conditions of low mucosal DNA in the 8.4-/5.5-kb DNA polymorphic band re- 6149 Downloaded from cancerres.aacrjournals.org on October 9, 2021. © 1990 American Association for Cancer Research. ODC. GENE MAPPING, AND COLORECTAL NEOPLASIA morphic region and a new 6.6-kilobase band appeared, suggest 12 3 4 M H ing that the chromosomal region encoding the 4.6-kilobase band found in the normal tissue may have been altered in the tumor yielding the 6.6-kilobase band. DNA from this tumor Kmm and adjacent mucosa samples was also digested with Hindlll, Xbal, EcoRl, BamHl, and Hinfl, but the restriction fragment length polymorphisms and the rearrangement in the tumor were not revealed using these restriction enzymes. mRNA Expression. ODC mRNA expression was examined in 18 patients with paired samples of colorectal carcinoma and adjacent, pathologically normal mucosa and in 4 patients with polyps—2 of whom had 2 polyps. It was found that the mRNA -5.5kb expression of the housekeeping enzyme TPI closely followed the amount of 28S and 18S RNA loaded into each well, and therefore this uniform expression between normal and tumor tissue could be used as an internal control by which expression 4.0 of ODC could be quantitated. Fig. 6 shows mRNA expression of ODC and TPI in 4 patients. TPI expression remained relatively constant between tumor and normal, whereas ODC showed increased expression in all tumors and polyps exam ined. In all samples, the ODC message was of constant size: 2.2 kb, and only 1 transcript was seen by Northern analysis. Of the 18 colorectal tumors (1 of which was a local recur rence), 7 were located in the rectum, 6 in descending colon or sigmoid, 2 in the transverse, and 3 in the cecum; 12 were moderately differentiated, 3 poorly, and 3 well differentiated. Six were Stage BI, 3 were Stage BII, 5 were Stage CI, and 4 were Stage CII. Polyps were tubular or \ ¡lionswith mild-to- moderate atypia. RNA was expressed as an ODC/TPI ratio for tumor and adjacent normal tissue. The fold increase of tumor compared with adjacent mucosa could therefore be derived. There was a significantly higher ODC mRNA expression in primary tumors than in adjacent mucosa (P = 0.001, with a Spearman correla tion coefficient of 0.79; and P < 0.001 for the Wilcoxon Hind m LOW STRINGENCY matched-pairs test). This is graphically demonstrated in Fig. Fig. 4. Southern hybridization of cDNA probe pODC10/2H to Hindl\\ digests of human (//), mouse (A/), and somatic cell hybrid DNA (Lanes 1-4) under low 1A. A similar finding was noted for polyps, mRNA expression stringency wash conditions. Lane I is positive for the 4.0-kilobase band, and always being higher than in normal tissue (Fig. 1A). Of 17 Lanes 1, 2, and 3 are positive for the 5.5-kilobase band. colorectal tumor primaries and one local recurrence, the fold increase of ODC mRNA expression in tumor compared with gion (Fig. 5). The pathologically normal adjacent mucosa of adjacent mucosa ranged from 1.29 to 12.2 with a median of this patient revealed an apparent new rare alÃeleof the poly 3.42 and a mean of 4.19 (±2.756). For 6 polyps, the fold morphism described above (Fig. 3). The band for this apparent increase ranged from 1.5 to 9.05 with a median of 2.8 and mean new alÃelecomigrated with the original, faint 4.6-kilobase band of 3.2 (±1.67). (Fig. 5, Lane A). This patient was, therefore, designated an 8.4- RNA expression and fold increase were examined with re /4.6-kb hétérozygoteatthis locus as compared with the 8.4-/ spect to site of tumor (rectum versus elsewhere in the colon), 5.5-kilobase hétérozygoteinthe adjacent control lane and the stage of disease (B versus C), and degree of differentiation. ODC 8.4-kilobase homozygote in the far right control lane. The mRNA expression was independent of these parameters (data patient's tumor retained the 8.4-kilobase alÃelein the poly not shown). Table 2 Distribution of ODC sequences with human chromosomes in Hindi 11digested human-mouse cell hybrid DNA Distribution of human ODC sequences in cell hybrids digested with Hindlll. The 5.5-kilobase band mapped to chromosome 2; the 4.0-kilobase band mapped to chromosome 7. Cell hybrids JSR-17S and VTL-7. retaining different regions of chromosome 7. localize the 4.0-kilobase band to the 7q31-qter region of chromosome 7. Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X 5.5-kilobasebandConcordant hybridsDiscordantno. of hybridsr; no. of Discordancy4.0-kilobase bandConcordant hybridsDiscordantno. of hybrids% no. of Discordancy276182582435002312342012371814442510292312342213372213372872024113122113333002114402312342112362013391816472212352212352592622133720IS432510292114401619542213372311322113382213372411311618531915442114401916462510292782318174920154316195414216020123718144418933141348 6150 Downloaded from cancerres.aacrjournals.org on October 9, 2021. © 1990 American Association for Cancer Research. ODC. GENE MAPPING. AND COLORECTAL NEOPLASIA A. CC15 ,j? _< Kb T A C?V ^ ¿ o o a. 1.0 U Q O Tumor Normal Pol») Polyp Fig. 7. A. Comparison of ODC mRNA expression in colorectal tumors and matched adjacent normal mucosa, and benign polyps and adjacent normal mu cosa. One patient had all 3 specimens of tissue analyzed. ODC and TPI expression were quantified using scanning densitometry and the ratios calculated. B, Com 2.8— parison of ODC activity (pmol CO2/h/mg protein) in colorectal tumors and adjacent normal mucosa and benign polyps and adjacent normal mucosa. Activity data are available for 15 of 18 tumor and mucosa samples (A) and 4 of 6 polyps and mucosa samples. 2.1-«* f- 0.20r-Ã’Q2 15C01 PstI '-oXLU^ce ODC Fig. 5. Southern hybridization of cDNA probe pODC10/2H to Psl\ digests of 0.5Ë0EXOXxxxx—X colorectal carcinoma CCI5 (7")run alongside DNA from normal adjacent patient Xx xI (A). A band is seen in Lane T measuring 6.6 kilobases. 00 05 10 15 2.0 25 Tumor ODC Activity (pmol CO/hr/mg protein) Fig. 8. Scattergram showing tumor mRNA expression venus ODC activity in the same colorectal tumor sample. O = local recurrence: all other measurements (x) for primary. No statistical correlation was found. TATATAT AM In 15 patients, paired samples from the same patient were analyzed for both ODC activity and mRNA expression to -28S investigate whether a direct correlation was present. Although all samples exhibited a significant increase in both ODC mRNA levels and ODC activity when compared with adjacent normal tissue (see Fig. IB), no correlation was found between ODC ODC RNA expression and activity within the same tissue samples -I8S (Spearman correlation coefficient 0.068, P = 0.81) (Fig. 8). DISCUSSION Human ODC gene sequences have been mapped previously to 2 pter —»p23and 7 cen —»qter(17). We have further localized ODC1 to 2p25 and ODC2 to 7q31 —»qter.Alhonen- Hongisto et al. (37) have found amplification of the chromo some 2 locus in human myeloma cells resistant to DFMO, suggesting but not proving that the locus of 2 is the functional Fig. 6. Northern blot of glyoxal gel analysis of tumor ( 7")and adjacent normal gene. We have compared the ODC mRNA expression in hu mucosa (A) of 4 patients with colorectal carcinoma. Lane M represents normal man-mouse hybrids containing human chromosome 2 but not mucosa from a patient who did not have colon cancer. The ODC (2.2 kilobases) 7, and chromosome 7 alone grown under similar conditions. and TPI hybridization signals are shown. ODC expression is markedly increased in tumors, whereas TPI remains relatively constant between tumor and adjacent Preliminary results suggest that the functional locus resides on mucosa. The position of 28S and 18S ribosomal bands are shown. chromosome 2 (data not shown) and that the locus on 7 is a 6151 Downloaded from cancerres.aacrjournals.org on October 9, 2021. © 1990 American Association for Cancer Research. ODC. GENE MAPPING. AND COLORECTAL NEOPLASIA pseudogene. The in situ hybridization results also indicate that The cooperation of the surgeons of the Surgical Developmental Oncol chromosome 2 may contain the major locus, since there was no ogy service, N. Petrelli, L. Herrera, J. Stulc, and A. Mittelman, is hybridization signal on chromosome 7. Fowler et al. (38) found gratefully acknowledged. Thanks also go to E. D. Holyoke for his that human-mouse cell hybrid methodology detected loci for a- encouragement and assistance. fucosidase on chromosomes 1 and 2, but in situ revealed only the functional locus on chromosome 1. In situ methodology REFERENCES therefore may not be as sensitive as the hybrid panel method, accounting for the lack of an ODC signal on chromosome 7. Pegg, A. E., and McCann. P. P. Polyamine metabolism and function. Am. J. Physiol.. 243:C2\2-22\, 1982. Interestingly, using the restriction enzyme Pstl, a restriction Heby, O. Role of polyamines in the control of cell proliferation and differ fragment length polymorphism was detected on chromosome entiation. Differentiation. 19: 1-20. 1981. 2. Two common alÃelesrepresented by 8.4- and 5.5-kilobase Janne. J.. Poso, H., and Raina. A. Polyamines in rapid growth and cancer. Biochim. Biophys. Acta, 473: 241-293. 1978. bands represent markers that will be useful in gene mapping Takano. S.. Matsushima, M., Erturk, G.. and Bryan. G. T. Early induction family studies and for determining the association and expres of rat colonie epithelial ornithine and .S'-adeno.syl-i-melhioninc decarboxylase activities by A'-methyl-A'-nitro-A'-nitrosoguanidine or bile salts. Cancer Res., sion of specific ODC alÃeleswith tumors and for possibly 41: 624-628, 1981. predicting a tumor phenotype in families. Hickok et al. (18) Rohzin, J., Wilson, P. S., Bull. A. W., and Migro, N. D. 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