Nucleic Acid Metabolism in Proliferating and Differentiating Colonie Cells of Man and in Neoplastic Lesions of the Colon1

[CANCER RESEARCH 31, 463-467, April 1971)

Frank Troncale, Ralph Hertz, and Martin Lipkin Departments of Medicine, The New York Hospital, Memorial Hospital for Cancer and Allied Diseases, and the Cornell University Medical College Â¡F.T.,M. L.Â¡,and the Department of Surgery, Memorial Hospital for Cancer and Allied Diseases, [R. H.J, New York, New York 10021

SUMMARY metabolic regulatory controls that lead to the onset of DNA synthesis and its cessation during the differentiation of normal Enzymes involved in the metabolism of nucleic acid intestinal cells have not been well defined. In small intestinal precursors were assayed in proliferating and maturing cells in cells of rodents, it has recently been shown that, as DNA the colon of man and in cells removed from polypoid lesions synthesis stops, marked changes develop in the activities of of the colon. Cells were separated from superficial and deeper enzymes that have a role in the synthesis of nucleotide layers of colonie mucosa by a recently developed precursors of DNA and RNA (13, 16). Differences in the tissue-planing instrument. Gradients of thymidine kinase, stability and turnover of these enzymes and their templates are thymidine phosphorylase, and adenine and hypoxanthine also present during the differentiation and migration of rodent phosphoribosyltransferase activities were found to characterize small intestinal cells, and it has been suggested that these different stages of cell differentiation in normal colon. factors may have a role in the differentiation of the cells (17). Thymidine kinase and phosphorylase were highest in young, These characteristics of differentiating intestinal cells have not proliferating cells and decreased during differentiation and been studied in man, either in normal or diseased states. migration of the cells to the mucosal surface. In this study, we have begun to explore these properties of Phosphoribosyltransferase activities were lowest in young, intestinal cells in man, and we have measured the activities of proliferating cells and increased during cell differentiation. In enzymes involved in nucleic acid metabolism in proliferating the polypoid lesions including carcinomas patterns of enzyme and differentiating colonie epithelial cells, as they migrate to activity characterizing young, proliferative cells were found. different levels of the colonie crypts. Several experiments were carried out: (a) the location in the colonie crypts of proliferating and nonproliferating epithelial cells was studied INTRODUCTION after pulse injection of TdR-3H,2 (mucosal biopsies and microautoradiography); (b) enzyme activities were studied in The mucosal lining of the gastrointestinal tract of man is proliferative and nonproliferative cells removed separately continuously replaced by epithelial cells that migrate from the deep portion of the mucosal crypts to the surface. Most of from different layers of normal colonie mucosa obtained at these cells have a life-span of 2 to 4 days and are rapidly operation; (c) enzyme activities were studied in cells lining the surfaces of polypoid lesions of the colon, some of which are extruded from the surface of the mucosa into the lumen of the believed to have an increased susceptibility to development of intestine (4, 23, 30). During migration, the epithelial cells carcinoma (22, 27). undergo rapid morphological and biochemical changes as they differentiate into cells that carry out mature functional activities in each region of the intestine. These include the MATERIALS AND METHODS cessation of DNA synthesis and proliferative activity. How ever, in areas of colonie mucosa near hyperplasias and Microautoradiographic Location of Proliferating and polypoid lesions and in the lesions themselves, epithelial cells Nonproliferating Cells. Two patients each were given injections are present that continue to synthesize DNA and proliferate of 10 mCi of TdR-3H. Both patients had inoperable throughout their entire life-span, as they migrate to the surface carcinomas of the colon with metastic lesions. Biopsies of of the mucosa (10). colonie mucosa were taken from a colostomy opening in In mammalian cells, factors that are believed to have a role Patient 1 and from the rectal mucosa of Patient 2, 1 and 2 hr, in DNA synthesis and proliferative activity include protein and respectively, after injection of TdR-3H. Microautoradiographs histone synthesis (5, 11, 31), ribosomal content (19), were prepared, and the location of cells incorporating TdR-3 H membrane potential (3), and cell mass (20). However, in the colonie crypts was determined microscopically (25). Preparation of Specimens of Colonie Mucosa for Enzyme 1This project was supported by NIH Grants and Awards 5 F03 AM Assay. Other specimens of histologically normal colonie 44662, CA-08921, and K 3-AM4468 from the USPHS. Received September 8, 1970; accepted December 14, 1970. 2The abbreviation used is: TdR-3H, thymidine-methyl-3 H.

APRIL 1971 463

mucosa were removed at operation from colons that contained The reaction mixture contained 150 mamÃ³les of a variety of different colon lesions: carcinomas, diverticular adenine-8-14C (specific activity, 200 cpm/mpmole), 50 disease, a lipoma, and adenomatous polyps. Strips of normal ni/miÃ³les of 5-phospho-D-ribosylpyrophosphate, 1 jmiole of mucosa 1 inch wide and 2 to 3 inches long were taken. The MgCl2, 10 /Â¿molesof Tris-HCl buffer (pH 8.0), and 0.1 ml of strips were always located 2 to 3 inches away from the lesion. supernatant containing enzyme, in a total volume of 0.2 ml. The reaction mixture was incubated at 37Â°for 30 min, and the Specimens were quickly placed on ice and transported to a cold room maintained at 32Â°F. The mucosa was separated reaction was stopped by immersion in boiling water. After from underlying tissues by careful dissection with scissors, laid cooling, the mixture was centrifuged at low speed for 5 min, out flat on a dissection board, cut into strips 1 cm wide and 6 and an aliquot of the supernatant was spotted on to 10 cm long, and then stretched and clamped tight on the diethylaminoethyl paper and treated according to the method platform of a recently developed tissue-planing apparatus (16). of Breitman (6). This instrument contains a razor mounted on a movable Hypoxanthine Phosphoribosyltransferase Assay (IMP: overhead housing. The blade quickly moves across the fresh Pyrophosphate Phosphoribosyltransferase, EC 2.4.2.8). The mucosa and removes progressively deeper layers of mucosa. A reaction mixture and assay method were the same as above micrometer attachment regulated the depth of each plane that except for the use of 150 m/imoles of hypoxanthine-8-'4C was cut. By this technique, the mucosa was separated into 3 (specific activity, 200 cpm/m/L/mole) in place of labeled approximately equal layers that were histologically identified adenine. as upper, middle, and lower thirds of the colonie crypts. Data on enzyme activities in upper, middle, and lower Specimens of colonie mucosa were also obtained from regions of colonie crypts obtained from normal strips of patients at proctoscopy by gently scraping the surface of colon mucosa were subjected to the following statistical analysis. with a modified surgical spoon curet. Mucosal trauma was The numerical values for enzyme activity in each region of the minimal, and histological examination of the scrapings crypt were correlated with the average distance of the cells in revealed sheets of surface mucosal cells, in some instances each region from the bottom of the crypt. The average cell attached to the upper portion of the crypts. The surface distance in each of the 3 regions was taken from data on epithelium of adenomatous polyps, villous adenomas, and histological slides. Data on enzyme activities from polyps, carcinomas was also gently scraped off with a No. 15 villous adenomas, carcinomas, and surface mucosa were Bard-Parker knife from specimens obtained either at surgery or subjected to an analysis of variance to bring out differences through the proctoscope. Hyperplastic polyps around 5 mm in among the various groups. size were homogenized completely. The tissue removed was homogenized in ice-cold Tris-HCl (pH 7.4) and adjusted to a RESULTS volume of 0.5 ml. Homogenates were centrifuged in the cold at 15,000 X g for 15 min; the supernatant was used for Microautoradiographic Measurements. The fraction of cells enzyme assay as reported previously (16), and precipitates incorporating TdR-3 H into DNA at each cell position in the were assayed for DNA. In the specimens of mucosa removed colonie crypts is shown in Chart 1. Most cells that were by the razor planing apparatus, the total amount of DNA synthesizing DNA were located in the lower third of the present averaged 0.19 mg; and in the specimens removed by surface scraping, the amount averaged 0.22 mg. Enzyme activities increased linearly during the time of incubation. Thymidine Rimise Assay (ATP:Thymidine S'-Phospho- transferase, EC 2.7.1.21). Thymidine kinase was assayed with the reaction mixture of Behki and Morgan (2) and the diethylaminoethyl paper method of Breitman (6). The 0.5 M Tris-HCl buffer (pH 8.0 at 37Â°)contained NaF, 1 mg/ml. Thymidine Phosphorylase Assay (Thymidine: Orthophosphate Deoxyribosyltransferase, EC 2.4.2.4). The reaction mixture contained 1 pinole of thymidine-2-14C (specific activity, 10s cpm/|/mole), 20 AmÃ³les of phosphate buffer (pH 7.5), and 0.1 ml of supernatant containing enzyme in a total volume of 0.5 ml. Incubation was carried out for 30 min at 37Â°.The reaction was stopped by immersion in boiling water, and 50 jul of supernatant were spotted on Whatman No. 3MM Bottom 0 0.2 0.4 0.6 0 0.2 0.4 Chromatographie paper strips. The end product of the Fraction of cells labeledwith TdR- reaction, thymine, was separated from thymidine in a decending Chromatographie system with the use of ethyl Chart 1. Changes in the fraction of cells labeled at each cell position in microautoradiographs of the colonie crypts. Patient 1 (left), 1 hr ace tate :H2O :formic acid (12:7:1) (upper layer used as after injection of TdR-3H, and Patient 2 (right), 2 hr after injection of solvent) (12). TdR-3 H. â€” at cell positions 30 and 60, boundaries between the upper, Adenine Phosphoribosyl transferase Assay middle, and lower thirds of the crypts. Very few cells in the upper third (AMP:Pyrophosphate Phosphoribosyltransferase, EC 2.4.2.7). are synthesizing DNA.

464 CANCER RESEARCH VOL. 31

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1971 American Association for Cancer Research. Nucleic Acid Metabolism in Human Colon crypts, below cell position 30. The number of DNA-synthesizing cells gradually decreased in the midregion of the crypts from about cell position 30 to cell position 60. Few E cells synthesized DNA as they reached the upper third of the S crypts, and none were synthesizing by the time they reached the surface. Similar spatial distributions of cells in DNA = 3 synthesis in colonie crypts have been found in other studies 'S 3 (23, 24). These cells migrate rapidly from the deeper crypt Â£ _o C regions to the surface, from where they are extruded into the 2: 2 E 2 intestinal lumen. Enzyme Activities in Regions of Normal Colonie Crypt. The comparative amounts of enzyme activity in the upper, middle, and lower thirds of the colonie crypts in normal mucosa are shown in Chart 2. There is a 4-fold decrease in the activity of 1121 lili thymidine kinase (12 colons) in the upper compared to the Chart 2. Change in activity (mean Â±S.E.)of 4 enzymes of purine and lower third of the crypts, indicating that the level of pyrimidine biosynthesis in human colonie crypts measured when the thymidine kinase falls off very rapidly during the migration of crypts were separated into lower, middle, and upper third by a razor the cells through the middle third of the crypts. Since these planning instrument. Note differing magnitude of the first ordinales. cells migrate at a velocity of 1 to 2 cell positions/hour (26) the The number of colon specimens studied is in parentheses, a, thymidine half-life of thymidine kinase is in the order of hours. Some kinase; b, adenine phosphoribosyltransferase; c, hypoxanthine phos enzyme activity is present in the upper third of the crypts, an phoribosyltransferase; d, thymidine phosphorylase. area where virtually all cells have stopped making DNA. Chart 20 shows a 4-fold increase in adenine colon than in the other tissues (0.025 >p> 0.01). Activity of phosphoribosyltransferase activity as the cells migrated from this enzyme progressively decreased in the cells of polyps of the lower to upper third of the crypts. The activity of this increasing size. In villous adenomas and carcinomas, levels of enzyme increases rapidly as cells stop proliferating in the activity reached those found in the immature proliferative cells midregion of the crypts. Hypoxanthine of normal colonie tissue. phosphoribosyltransferase activity also increased during The activity of hypoxanthine phosphoribosyltransferase was migration of the cells to the surface area of mucosa as seen in significantly higher in mature colon cells and small Chart 1c. However, this increase was not as marked as seen hyperplastic polyps (0.05 > p > 0.025) than the other lesions with adenine phosphoribosyltransferase. A decrease in the and decreased to reach the low levels found in immature activity of thymidine phosphorylase was observed as cells proliferative cells (Chart 3c). However, a similar activity migrated from the middle to the upper third of the colonie gradient was not found with thymidine phosphorylase; the crypts (Chart 2d). amount of activity in hyperplastic tissue appeared to be closer The decrease in thymidine kinase and increase in adenine to those found in proliferative than in mature cells (Chart 3d). phosphoribosyltransferase activities were significantly correlated with cell distance from the bottoni of the crypt (0.05 > p > 0.01), and for hypoxan thine DISCUSSION phosphoribosyltransferase the increase in activity was borderline significant (0.1 > p > 0.05). A linear increase or Previous work has shown that the mean generation time of decrease in thymidine phosphorylase activity was not actively proliferating colonie epithelial cells in man is about 2 observed. days. In the proliferative region, approximately 30% of Enzyme Activities in Polypoid Lesions. In Chart 3, epithelial cells are making DNA, and others are moving comparative enzyme activities expressed per mg of DNA are through one of the phases of the proliferative cell cycle. Under shown in cells removed in vivo from the surface of the colonie normal conditions, most migrating epithelial cells in the colon mucosa and from the surface of the lesions. Cells were of man stop making DNA 12 or more hr before they reach the removed from the mature nonproliferative zone of normal luminal surface of the intestine, a situation analogous to small mucosa; small hyperplastic polyps less than 1 cm in diameter; intestine when cells reach the upper region of the crypts and large polyps greater than 1 cm in diameter, most of which begin to move onto the villi (28). As cells migrate through the were adenomatous; villous adenomas; and carcinomas. Data on midregion of the crypts of both small intestine and colon, enzyme activities in lower-third proliferative cells shown in although progressively fewer cells replicate, they are still Chart 2 are replotted asÂ¿ for comparison. capable of being recalled into the proliferative cell cycle and of As shown in Chart 3a, thymidine kinase activity was making new DNA, if new cells are needed, as for example after significantly greater in villous adenoma and carcinoma cells radiation damage (8); these cells are in a "transitional" stage than in the other specimens (p < 0.005) and approximated between the proliferative and mature phases of their life cycle levels of activity found in proliferative cells of normal colonie (28). However, once they have differentiated sufficiently to tissue. reach the colonie crypt surfaces or migrate onto the villi, they Adenine phosphoribosyltransferase activity (Chart 3b) was normally are no longer able to proliferate. significantly greater in the mature surface cells of normal This cessation of DNA synthesis and proliferative activity is

APRIL 1971 465

epithelial cells. Thymidine kinase, while present in young, proliferating cells located in the crypts, was not present in villous cells. This enzyme is widespread in proliferating cells and is absent from only a few cell types (9). Previous studies have shown that the half-life of thymidine kinase is characteristically in the order of minutes to a few hours (9), and in small intestine and liver of rat it is 2.6 hr (7, 16). Enzyme activity is increased in rapidly growing tumors (15) and in proliferating tissue culture cells (21). In the present study, thymidine phosphorylase activity did not increase during migration and differentiation of colonie cells of man. This is in contrast to the findings in small intestine, where a marked increase was observed during cell migration (16). This increase, together with the decrease in enzyme activity observed in leukemic cells (14, 29), had suggested that this enzyme might be involved in the regulatory control of DNA synthesis by limiting the availability of

Â«e 0 thymidine (16). In this instance, it is not known whether the (231 (U) (8l 12) (6l (111 failure to detect an increase in thymidine phosphorylase

5Z activity during the migration of colonie cells might be connected with their susceptibility to continued DNA AOrS synthesis and mitosis. Thymidine synthetase activity, not measured in this study, could reflect DNA synthesis more 3oE'S directly than either thymidine kinase or thymidine phosphorylase. 2ESi In the colon, as in small intestine, adenine and hypoxanthine phosphoribosyltransferase activities increased 1st= with normal differentiation and migration of the cells. However, in the colon, the magnitude of the adenine 161"|,9liÃ(2011101 (71 (21 phosphoribosyltransferase increase was greater than hypoxanthine phosphoribosyltransferase, in contrast to the 'EThymine findings in small intestine (16). In contributing to the formed, formation of AMP's and IMP's, these enzymes may make it limÃ³les/mgDNA/30-â€”-Â©fin(19) possible for salvage pathways to reclaim nucleic acid precursor materials from the intestine. The question of why levels of various enzymes change during differentiation of normal cells and the possible significance of these changes has received attention in recent years. In intestinal cells, which undergo rapid differentiation (91 (91 121 6l and move into new environments within hours, the rapid M SP LP VA CA181i(71L increase or decrease in the activity of these enzymes appears to Chart 3. Change in enzyme activity (mean Â±S.E.) in cells removed be influenced by differential rates of turnover of both the from normal colon and from neoplastic lesions. M, cells scraped by enzymes and their templates. These have been postulated to spoon curet from the surface of normal mucosa; SP, small polyps less than 1 cm; LP, surface cells scraped from polyps 1 cm or larger in act as regulatory controls contributing to the normal diameter, VA, from villous adenomas; CA, from carcinomas. L, lower- differentiation of the intestinal cells (17). third proliferated cells of normal mucosa from Chart 2, shown here The rapid appearance or disappearance of metabolic for comparison. Numbers in parentheses refer to the number of speci activities and the degradation of some of these enzymes could mens studied, a, b, c, and d refer to same enzymes as in Chart 2. also involve active synthetic processes, as suggested by experiments in other systems. For example, the degradation of tyrosine aminotransferase (1, 18) and glucose 6-phosphate accompanied by the rapid development of morphological and dehydrogenase (32) may be increased by the synthesis of biochemical features that prepare the cells to function as specific proteins. However, it is not known whether this type elements lining the surface of the gastrointestinal tract. In this of regulatory activity might contribute to changes in enzyme study, thymidine kinase activity was high in proliferative crypt activity in normal differentiating intestinal cells or in the cells cells and declined as the cells approached the surface of the of these neoplastic growths. The present study shows that, in mucosa. Recently, it was shown in small intestine of the rat the cells of these neoplastic growths, "juvenile" patterns of (13, 16) that the levels of several of the enzymes involved in enzyme activity that are found in proliferative cells persist, the synthesis and degradation of nucleic acid precursors and patterns of activity that biochemically identify change very rapidly during the normal differentiation of the well-differentiated cells do not develop.

466 CANCER RESEARCH VOL. 31

ACKNOWLEDGMENTS Comparative Study of the Thymidine Kinase and Thymidylate Kinase Activities and of the Feedback Inhibition of Thymidine We thank Dr. M. E. Balis for helpful suggestions and criticisms and Kinase in Normal and Neoplastic Human Tissue. Cancer Res., 28: Dr. Melvin Schwartz for aiding the statistical analysis. 2068-2077, 1968. Microautoradiographs were prepared by Dr. Eleanor Deschner, and 16. Imondi, A. R., Balis, M. E., and Lipkin. M. Changes in Enzyme technical assistance was provided by Miss Luba Geller. Levels Accompanying Differentiation of Intestinal Epithelial Cells. Exptl. Cell Res., 58: 323-330, 1969. 17. Imondi, A. R., Lipkin, M.. and Balis, M. E. Enzyme and Template Stability as Regulatory Mechanisms in Differentiating Intestinal REFERENCES Epithelial Cells. J. Biol. Chem., 245: 2194-2198, 1970. 18. Kenney, F. T. Turnover of Rat Liver Tyrosine Transaminase: 1. Aurricchio, F., Martin, D., and Tomkins, G. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1971 American Association for Cancer Research. Nucleic Acid Metabolism in Proliferating and Differentiating Colonic Cells of Man and in Neoplastic Lesions of the Colon

Frank Troncale, Ralph Hertz and Martin Lipkin

Cancer Res 1971;31:463-467.

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