and in Vitro by Calcium Inhibition of Human Colonic

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colonie epithelial cells in six of these patients showed a statistically ... The costs of publication of this article were defrayed in part by the payment ... Tissue Culture Procedures for in Vitro Calcium Modulation Studies. ... simpler method described in the paragraph above. ... characteristics are listed in Table 1, patients 1-9.
Inhibition of Human Colonic Epithelial Cell Proliferation in Vivo and in Vitro by Calcium Michel Buset, Martin Lipkin, Sidney Winawer, et al. Cancer Res 1986;46:5426-5430.

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(CANCER RESEARCH 46, 5426-5430, October 1986)

Inhibition of Human Colonie Epithelial Cell Proliferation in Vivo and in Vitro by Calcium1 Michel Buset, Martin Lipkin, Sidney Winawer, Shanti Swaroop, and Eileen Friedman2 Laboratory of Gastrointestinal Cancer Research and Gastroenterology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT Nine patients at high risk of developing colon cancer were placed on daily p.o. supplementation of 1500 mg of calcium for 4-8 weeks. The colonie epithelial cells in six of these patients showed a statistically significant decrease in their |3H|thymidine labeling indices in tissue culture so that they resembled those of patients at low risk of developing colon cancer. The three nonresponders had similar labeling indices before and after calcium supplementation. Biopsies from each of nine high-risk patients exhibited a decrease in proliferation when they were cultured in vitro with a high level of CaCl2 (2.2 HIM compared with the 0.1 IHM optimum value for proliferation). Two adenomas and two carcinomas showed a different pattern of response than normal cells, exhibiting no inhibition of growth at 2.2 HIMCaCl2. These data indicate that the growth inhibition induced by high levels of extracellular calcium levels is lost at a stage in tumor development before cells become malignant.

INTRODUCTION Enhanced epithelial cell proliferation in patients at increased risk of developing colon cancer reflects an expansion of the proliferative compartment in their colonie crypts. A more quies cent epithelium is found in patients at lower risk of developing colon cancer (1-3). In a previous study from one of us (4), 10 patients placed on dietary calcium supplementation showed an average decrease in colonie epithelial cell proliferation as as sayed by [-'H]dThd3 labeling of colonie biopsies in an organ culture system. Therefore, reversion of this biomarker associ ated with increased risk suggested that dietary calcium was potentially an effective means of intervention. To study this phenomenon more directly we decided to utilize a more readily manipulatable system, tissue culture of the colonie epithelial cells. In organ culture a colonie biopsy is placed directly into culture medium without digestion, pulsed with ['HJdThd for 1 h; then the tissue is formalin fixed, very carefully sectioned so that the entire test tube-like crypt from the base to the top (about 50 cells long) is contained within one section, and processed for autoradiography. In tissue culture, in contrast, the epithelial cells simply are cultured out from the partially digested colonie epithelial layer as patch cultures in the presence of ['HJdThd, fixed, directly coated with emulsion, and scored under a microscope. In this tissue culture method, as in organ culture, the high-risk and low-risk subjects have been distinguished. Individuals at increased risk for the devel opment of colon cancer generally have a higher overall labeling index than patients at lower risk. In an earlier study (5), labeling indices were determined for 11 subjects symptomatic with either adenomas or carcinomas, 15 subjects at increased risk defined Received 3/4/86; revised 7/1/86; accepted 7/3/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by National Cancer Institute Grant 28822 from the National Large Bowel Cancer Project to E. F., National Cancer Institute Grant 40876 and American Cancer Society Grant SIG-7 to M. L., Institutional Grant CA 08748, and grants from the Fondation Rose et Jean Hoguet. Brussels, Belgium, and the North Atlantic Treaty Organization to M. B. 2 To whom requests for reprints should be addressed. 3The abbreviations used are: dThd, thymidine; LI, labeling index(es).

by a positive family history of colon cancer but asymptomatic, and 5 subjects from families free of colon cancer for 2 or more generations. Their mean [3H]dThd LI were, respectively, 12.2, 17.2, and 5.7%, although occasionally a high-risk patient would have a LI less than 10%. Therefore, this simple measurement reflects an abnormal excess proliferation which can be seen in patients at increased risk of developing colon cancer and is conspicuously absent in individuals at low risk. MATERIALS AND METHODS Tissue Culture Procedures for Biopsies from Patients on Calcium Intervention Study. Colonie mucosal biopsies were cultured on gelatin films in NCTC medium supplemented with 15% fetal calf serum and 2.2 mM CaCl2 exactly as detailed (5), with the exception that the pentagastrin concentration utilized was 5 Mg/ml. All the data in Tables 2-4 were obtained by this method. These partial digests were placed on gelatin films in 0.2 ml of medium and allowed to attach for 45 min at 37°Cin a CO2 incubator; then 1 ml of medium containing a lethal level of pHjdThd (5 ^Ci/ini) was added and the cells were cultured for 24 h. The very high level of ['HjdThd ensured that each cell would pass through S phase only once during the labeling period and then be held in late S-G2 as the badly damaged chromatin could not condense and undergo mitosis. During this time the colonie epithelial cells of the digest migrated from the attached expiant to form a flat patch on the surface of the Petri dish. The monolayer cells were then fixed twice with 5 ml methanol/dish, air dried, coated with undiluted NTB-2 Kodak emulsion, exposed for 10 days, developed, and counterstained with hematoxylin. All cells in each epithelial patch colony were read by microscopy. Tissue Culture Procedures for in Vitro Calcium Modulation Studies. Each biopsy was very finely minced so that no large pieces (>1 mm) remained, and placed into 3 ml of antibiotic-containing wash medium (6) plus 1 ml each of hyaluronidase, neuraminidase, and collagenase (5). Colonie bacteria often adhered to mucus secreted by the epithelial cells, so an addition was made of 0.1 ml of 20% Mueomyst (MeadJohnson), a solution of acetylcysteine. Digestion occurred at room temperature on a blood rotator, so the digests were continuously gently shaken for 3-5 h to remove mucus, instead of the 1 h used in the simpler method described in the paragraph above. The digest was plated in serum-free NCTC 168 medium supplemented with transferrin, in sulin, hydrocortisone, insulin, pentagastrin, sodium deoxycholate, epi dermal growth factor, and selenous acid as described (5), but also contained 3 x 10~5 M isobutylmethyl xanthine, and 10~4 M each of ethanolamine and phosphoethanolamine. The digests consisted of groups of epithelial cells and partly digested colonie crypts. A substrate consisting of 10 ¿tg of fibronectin (Bethesda Research Laboratories), 10 ¿tg of collagen I (Vitrogel), and 30 ¿