Cholesterol Sulfate Accumulation in Tumorigenic

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[CANCER RESEARCH 48, 5289-5295, September 15, 1988] Cholesterol Sulfate Accumulation in Tumorigenic and Nontumorigenic Rat Esophageal Epithelial Cells: Evidence for Defective Differentiation Control in Tumorigenic Cells1

J. I. Rearick, G. D. Stoner, M. A. George, and A. M. Jetten2

Department of Biochemistry; Kirksville College of OstÃ©opathieMedicine, Kirksville, Missouri 63501 fJ. I. R.J; Department of Pathology; Medical College of Ohio, Toledo, Ohio 43699 [G. D. S.J; and Cell Biology Group, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [M. A. G., A. M. JJ

ABSTRACT the effects of two modulators of this pathway of differentiation: calcium ions and retinoic acid. Increasing calcium ion concen In this study the regulation of squamous cell differentiation in several tration in the medium has been shown to stimulate squamous rat esophageal epithelial cell lines is examined. Nontumorigenic RE-149 cell differentiation in several in vitro systems (11, 12) including cells undergo a program of squamous cell differentiation at confluence. rat esophageal cells (9), whereas retinoids are potent inhibitors This program of differentiation is influenced by the concentration of of this differentiation process (13-15). In this paper the accu calcium in the medium and by the presence of retinoic acid. High calcium concentration stimulates terminal cell division, as indicated by a reduction mulation of cholesterol sulfate and the expression of steroid in colony-forming efficiency, and increases the expression of the differ sulfotransferase activity were examined during the differentia entiated phenotype as indicated by an increase in cholesterol sulfate tion process. Our findings indicate that tumorigenic cells ac accumulation and cross-linked envelope formation. Retinoic acid inhibits quire defects in the control of differentiation as indicated by squamous cell differentiation as both cholesterol sulfate accumulation altered responses to calcium and retinoic acid. and cross-linked envelope formation are reduced. Two tumorigenic cell lines, RE-B2 and RE-2BT, do not undergo squamous cell differentiation in vitro. High calcium concentration in the medium did not significantly MATERIALS AND METHODS reduce colony-forming efficiency or induce cross-linked envelope forma tion. High calcium concentration or retinoic acid had only a limited effect Culture Medium. All experiments with RE-149, RE-B2, and RE-2BT on the accumulation of cholesterol sulfate. RE-B2T cells exhibit high were carried out in REM-1 medium. This medium is a modification of levels of cholesterol sulfate and cholesterol sulfotransferase activity. PFMR-4 medium (16, 17) in which calcium was reduced from 1.0 to 0.1 HIMand contained 5 ng/ml of EGF,3 1 ^M hydrocortisone (Stera- These levels appear no longer controlled by calcium or retinoic acid, indicating that the synthesis of cholesterol sulfate occurs in a constitutive loids, Wilton, NH), 1 f<\reach of ethanolamine and phosphoethanola- manner. The altered responses of RE-2B and B2T cells to calcium and mine (Calbiochem-Behring, LaJolla, CA), and 5 mg/ml of dialyzed retinoic acid suggest that these malignant cells have acquired one or more fetal bovine serum protein (9). Gentamicin (Schering Corp., Kenil- defects in the control of differentiation. worth, NJ) was added to the medium at a concentration of 50 Mg/m'- Fetal bovine serum was purchased from Flow Laboratories, McLean, VA (Lot 29101084) and was selected from several commercial lots INTRODUCTION based upon its ability to stimulate the clonal growth of rat esophageal epithelial cells (9). Fetal bovine serum was dialyzed as described by In certain regions of the world the esophagus is a major site Kaighn et al. (18), and the protein content of the dialyzed serum was for cancer, and esophageal cancer is a major cause of death (1, determined by the method of Lowry et al. (19). Colony-forming effi 2). Dietary habits and both tobacco and alcohol consumption ciency, defined as the percentage of plated cells able to form colonies, have been implicated as important risk factors for the devel was determined as described previously (20). opment of esophageal cancer (1-5). Our laboratory (G. D. S.) Derivation of Cell Lines. Esophagi were obtained from 4- to 6-wk- has been involved in the development of in vitro systems to old F344 rats. Methods used for collection of the esophagi, progressive enzymatic dissociation of the epithelium, and initiation of primary investigate the effects of carcinogens on rat esophageal epithe cultures were as described previously (9). Cell line RE-149 is an im lial cells (6-8). Culture conditions for the serial propagation of mortalized cell line derived "spontaneously" from primary cultures of normal rat esophageal cells have been developed (9), and cells normal rat esophageal cells. Cell line RE-B2 was derived from an have been transformed following exposure to the carcinogen, outgrowth of epithelial cells from an expiant of F344 rat esophagus jY-nitrobenzylmethylamine (7). Epithelial cell lines have been that had been treated in primary culture with 5 ^g per ml of NBMA. developed that, following in vivo transplantation into syngeneic The characteristics of this cell line have been described (7). Cell line rats, produce well-differentiated squamous cell carcinomas (6, RE-B2T was derived by enzymatic dissociation of a squamous cell 7). carcinoma produced within 7 mo after s.c. inoculation of RE-B2 cells The lining of the esophagus consists of a stratified, squamous into the intrascapular region of 1-day-old F344 rats. As indicated in Table l, RE-B2T cells have a higher colony-forming efficiency in cell epithelium. Since the transformation of normal cells is agarose (2.53 versus 0.04%) and are more strongly tumorigenic than usually associated with alterations in the control of proliferation RE-B2 cells. and differentiation (10), a major objective of studies in in vitro Assay of Cross-Linked Envelopes. For the determination of the carcinogenesis has been to identify the defects in these controls spontaneous formation of cross-linked envelopes, medium and cells that lead to a transformed phenotype. In this paper we compare were collected and centrifuged, and the pellet was resuspended in 20 the regulation of squamous cell differentiation in nontumori- mM dithiothreitol and 2% sodium dodecyl sulfate. After incubation for genic and tumorigenic esophageal epithelial cells and examine 5 min at 95Â°C,detergent-resistant envelopes were counted as described previously (20, 21). The same procedure was followed to measure the Received 2/26/88; revised 6/13/88; accepted 6/21/88. competence to form cross-linked envelopes except that cells were first The costs of publication of this article were defrayed in part by the payment treated with 25 jig/ml of Ca2+-ionophore Ro 2-2985 (Hoffmann-La of page charges. This article must therefore be hereby marked advertisement in Roche, Inc.) in the presence of 2 mM Ca2+. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1A portion of this research was supported by National Cancer Institute Grant CA 28950 to G. D. S. 3The abbreviations used are: EGF, epidermal growth factor; NBMA, N- 2To whom requests for reprints should be addressed. nitrosobenzylmethylamine. 5289

Assay of Cholesterol Sulfate. Cholesterol sulfate was determined as marized in Table 1. RE-149 is an immortal cell line which described previously (22). Briefly, cells were labeled with 20 to 40 fid/ ml of Na235SO4(carrier-free; ICN, Irvine. CA) for 24 h, harvested by exhibits many of the differentiation characteristics of normal rat esophageal cells (6-9). Since normal rat esophageal cells trypsinization, pelleted by centrifugation, and extracted with 4 ml of chloroformrmethanol (2:1). After the addition of 1 ml of 0.1 M KC1, are difficult to obtain in large quantities this cell line was chosen the extracts were vortexed and centrifuged, and the upper aqueous to examine several biochemical parameters of squamous differ phase was removed. The organic phase was extracted once more with entiation. The proliferation of RE-149 cells was dependent on 2 ml of CHjOH/0. l M KCI. The resulting organic phase was isolated the presence of both EGF and dialyzed fetal bovine serum in and taken to dryness, and radioactivity was determined. The lower the medium (9). These cells did not form colonies in soft agar phase was subjected to thin-layer chromatography using a solvent and were nontumorigenic in syngeneic hosts. In contrast, RE- system composed of CHCI.i:CH3OH:acetone:glacial acetic acid:HÂ¡O (8:2:4:2:1) and Silica Gel G plates (Analtech, Newark, DE). Steroids B2 cells, which were derived after NBMA treatment of primary were visualized as described previously (22). cultures, could grow in the absence of EGF or serum. These Assay for Sulfotransferase Activity. Cells, grown in 60-mm dishes, cells formed colonies in soft agar and were tumorigenic. The were collected by trypsinization, pelleted by centrifugation, and resus- RE-2BT cell line was established from one of the tumors pcnded in 50 min 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, induced by RE-2B cells and exhibited an enhanced ability to pH 7.3. The suspension was sonicated 3 times for 15 s using a W-225 form colonies in soft agar as well as increased tumorigenicity. sonicator (Ultrasonic Heat Systems). Sulfotransferase activity was measured by determining the incorporation of [35S]suIfate from 3'- As indicated, all these cell lines are aneuploid. phosphoadenosine-5'-phosphosulfate into cholesterol sulfate as de When RE-149 cells were grown to confluence, a pathway of scribed previously (23). squamous cell differentiation was induced as indicated by the Assay for Transglutaminase Activity. Transglutaminase activity was gradual increase in the formation of cross-linked envelopes. determined as described previously (20) by measuring the incorporation of ['Hjputrescine (18.6 Ci/mmol; DuPont, Boston, MA) into casein High calcium ion concentration in the medium greatly stimu lated this pathway of differentiation. Fig. \A shows the growth hydrolysate. Under all conditions tested more than 95% of the total of RE-149 cells grown in REM-1 medium containing either 0.1 transglutaminase activity was associated with the paniculate fraction. HIMor 1.0 HIMCa2+. High Ca2+ concentration caused a slight decrease in the exponential growth rate and a significant reduc RESULTS tion in saturation density. A comparison of the morphology of Morphological and Growth Properties of Esophageal Cells. the cells is shown in Fig. 2, A and B. RE-149 cells, grown in Several characteristics of the cells used in this study are sum- low Ca2+, formed cross-linked envelopes "spontaneously" only when maintained at confluence for longer than 3 days. High linesCellTable 1 Characteristics of rat esophageal epithelial cell Ca2+ concentration stimulated the formation of cross-linked envelopes. A quantitation of the cross-linked envelope forma and pendent growth in tion is shown in Fig. IB. RE-149 cells were competent to form EGF growth agarose (% of in syngeneic lineRE-149 dependence+KaryotypeAneuploid CPE")00.04 hosts0/25 cross-linked envelopes at all stages of the growth curve; this competence was not influenced by Ca2+. RE-B2 Aneuploid 14/25 Cholesterol Sulfate Synthesis in Esophageal Cells. Recently, RE-B2TSerum AneuploidAnchorage-inde2.53Tumorigenicity*28/29 " CFE, colony forming efficiency. we have established that the synthesis of cholesterol sulfate is " All three cell lines were tested for tumorigenic potential following s.c. a sensitive and convenient marker of squamous cell differentia inoculation into I-day-old syngeneic rats. There were 25 to 29 rats per cell line, tion (22, 24). To determine whether RE-149 cells synthesize and each rat received 2 to 3 x 10' cells. RE-149 cells were nontumorigenic in all cholesterol sulfate during squamous differentiation, cells grown 25 of 25 rats. RE-B2 cells produced squamous cell carcinomas in 14 of 25 rats within 7 lo IO mo. RE-B2T cells produced squamous cell carcinomas in 28 of 29 at confluence in the presence of low and high calcium were rats within 6 to 8 wk. incubated with ["SJsulfate for 24 h, harvested, and extracted

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TIME(doys) TIME (days) TIME (days) Fig. 1. Production of cholesterol [3!S]sulfate and cross-linked envelope formation as a function of the growth curve. RE-149 cells were grown in low (0.1 mM; O, A) and high (1.0 mM; â€¢A) <'-.<". and at the indicated times, cell number 1.11. cross-linked envelopes (B), and cholesterol ["SJsulfate accumulation Â«) were determined. The variation in the cholesterol sulfate determination between duplicates was 15% or less. In B, triangles represent the competence of cells to form cross- linked envelopes; the circles represent "spontaneous" formation of cross-linked envelopes.

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I* M^ â€¢â€¢â€¢ t Fig. 2. Morphology of RE- 149, RE-B2, and RE-B2T cells. .-<.confluent RE- 149 cells grown in 0.1 mM Ca2* (Day 4 of growth); B, confluent RE- 149 cells grown in 1.0 mM Ca2* (Day 4 of growth). Arrowheads indicate cross-linked envelope formation; C, confluent RE- 149 cells grown in the presence of 1.0 mM Ca2* and 2 x 10~7M retinoic acid: D, confluent RE-B2 cells grown in 0.1 mM Ca2*; E, confluent RE-B2 cells grown in 1.0 mM Ca2*; F, confluent RE-B2T cells grown in 0.1 mM Ca2*. with chloroform/methanol, and the resulting extract was par The production of cholesterol sulfate at different stages of the titioned against 0.1 M KC1. The resulting organic phase was growth curve is shown in Fig. 1C. Cultures of RE-149 cells subjected to thin-layer chromatography. The results (Fig. 3) grown in low calcium showed an increase in cholesterol sulfate show that most of the radioactivity comigrated with authentic after they reached confluence. The presence of high calcium cholesterol sulfate. In other studies we have characterized the greatly enhanced the rate of cholesterol sulfate accumulation. labeled product more rigorously and identified it as cholesterol- Under both low and high calcium conditions the increase in 3-sulfate (22). No qualitative differences in 35S-labeled lipids cholesterol sulfate preceded the formation of cross-linked en were identified between cells grown in low and high calcium. velopes. Fig. 4 shows the Ca2+ dependency of the increase in 5291

cholesterol sulfate accumulation. Cholesterol sulfate synthesis B increased with increasing Ca2+ concentrations and started to level off at concentrations greater than 1.0 mM Ca2+. m To examine the cause of the increase in cholesterol sulfate o by calcium, the levels of cholesterol sulfotransferase, the en zyme catalyzing the synthesis of cholesterol sulfate, were deter mined in RE-149 cells. Confluent cultures grown in high cal cium exhibited approximately 7-fold higher levels of cholesterol sulfotransferase than confluent cultures grown in low Ca2+ (Table 2). The differences in cholesterol sulfate accumulation appeared to be specific changes, since no significant differences in the incorporation of labeled sulfate into chloroform/metha- i nol-insoluble pellet (CM pellet; Table 3) were found. o Transglutaminase type I has functioned in several systems as i an indicator for squamous differentiation (12, 25, 26). However, RE-149 cells exhibited relatively high levels of transglutaminase activity at all stages of the growth curve (not shown) consistent 10 15 O 5 10 15 with the competence to form cross-linked envelopes at all stages (Fig. 1Ã„).High Ca2+concentrations increased transglutaminase DISTANCE MIGRATED (cm) Fig. 3. Thin-layer chromatography of ("SJsulfate-labeled organic-soluble ma activity by only 2-fold (Table 3). Under all conditions tested, terial from RE-149 cells grown at low (A) or high (fi) calcium ion concentrations. more than 95% of the transglutaminase activity was associated The arrows indicate the migration positions of the standards (1, cholesterol with the particulate fraction of homogenized RE-149 cells, sulfate: 2, cholesterol), which were chromatographed in the same lanes as the radioactivity. Only part of the total radioactivity was used for chromatography. suggesting that the activity consisted of predominantly type I (epidermal) transglutaminase. Action of Retinoic Acid. Retinoids have been reported to inhibit squamous differentiation in several cell systems (13- 15). Treatment of RE-149 cells with retinoic acid inhibited the spontaneous formation of cross-linked envelopes (Fig. 2C; Ta ble 3) and inhibited the accumulation of cholesterol sulfate in 80 the presence of either low or high Ca2+ concentrations. A UJ concentration dependency of the inhibition by retinoic acid is i shown in Fig. 5. Retinoic acid was active at concentrations as L 60 low as 10"" M; the 50% effective concentration was 5 x 10~'Â° M. Retinoic acid had little influence on the transglutaminase A activity of RE-149 cells; as in untreated cells all transgluta minase activity was associated with the particulate fraction. No increase in cytosolic transglutaminase (tissue transglutaminase) activity could be demonstrated. 20 Cholesterol Sulfate Synthesis in Tumorigenic Cells. The two tumorigenic cell lines RE-2B and RE-2BT did not undergo terminal differentiation either at confluence or in the presence of high calcium. Cultures of these transformed cells reached 0.4 0.8 1.2 1.6 higher cell densities at confluence than RE-149 cells (Fig. 2, D CALCIUM CONCENTRATION (mM) Fig. 4. Stimulation of cholesterol ["Sjsulfate synthesis as a function of the and F). High calcium concentration in the medium had little calcium concentration. RE-149 cells were plated at 10" cells/60-mm dish, and 2 effect on the rate of proliferation but did cause a significant days later they were changed to the indicated calcium concentration. After 4 more (approximately 40%) increase in the saturation density (not days the synthesis of cholesterol sulfate was determined. Medium was renewed shown). In addition, high calcium concentration did not cause every 24 h. The graph is representative for two independent experiments. a significant reduction in colony-forming efficiency (Table 3). Although both cell lines contained high particulate transgluta minase activity and were competent to produce cross-linked Table 2 Comparison of the levels of cholesterol sulfotransferase activity in rat envelopes (about 80 to 90% of cells formed envelopes as in RE- esophageal epithelial cells 149 cells), no "spontaneous" formation of cross-linked enve Cells were grown to confluence (Day 6 of culture) in the presence of 0.1 or 1.2 lopes was induced at confluence or in high calcium (Fig. 2, D mM < ;i (final concentration) and harvested, and cholesterol sulfotransferase and E; Table 3). High Ca2+ concentration increased the levels activity was determined. Sulfotransferase determinations varied 15% or less between duplicates. of cholesterol sulfate by 3-fold in RE-B2 cells; however, these sulfotransferase levels were 10-fold lower than in RE-149 cells. RE-B2T cells CelllineRE-149 (dpm/h/Mgprotein)40(1)Â° exhibited very high levels of cholesterol sulfate at all stages of mm Ca2* 1.2mMCa2*0. the growth curve (not shown); these levels were only slightly 274(6.9)2.5(1) influenced by either high Ca2+ or by the presence of retinoic RE-B2ConditionO.I 1 mM Ca2* acid (Table 3). The "S-labeled radioactivity in the organic phase 1.2mMCa2*Cholesterol 2.0 (0.8) of the extracts from both RE-B2 and RE-B2T cells comigrated RE-B2T 0.1 mMCa2* 1377 with cholesterol sulfate when analyzed by thin-layer chroma Â°Numbers between parentheses, fold increase. tography (not shown). The high levels of cholesterol sulfate in 5292

Table 3 Effect of calcium and retinole acid on the accumulation of cholesterol sulfate, transglutaminase activity, cross-linked envelope formation, and colony-forming efficiency in rat esophageal epithelial cells Cells were grown in the presence of either no additions or 1.0 mM Ca2* (final concentration) and/or 2 x 10~7 M retinoic acid. ["SJSulfate incorporation into cholesterol sulfate and into the chloroform/methanol-insoluble pellet (CM pellet), transglutaminase activity, cross-linked envelope formation, and colony-forming efficiency were determined during the exponential phase and 3 days after the cell reached confluence. Differences in duplicate values of the cholesterol sulfate determination were 15% or less. The transglutaminase activity is shown as total cellular activity. in [35S]sulfate CM pellet (dpm/ activity (cpm/h//jg CEÂ°formation(%)<0.52.0 CelllineRE-149Exp.ConO.RE-B2Exp.Confi.RE(dpm/^gprotein)2.7(1)"8.5(3.1)183(79.6)4.6(1.7)24.8titi protein)21520518720621729233932029630452747772Transglutaminaseprotein)65.0 efficiency(%)30.5

196]''79.3Â±3.3C Â±5.025.9 Â±5.0[97]144.4 Â±1.036.5 Â±3.93.1 Â±7.1[98]92.5 Â±4.7<0.53.0 Â±1.427.3 Â±4.2[97]101 Â±5.320.8 (9.2)5.1(1)7.2(1.4)22.6 Â±3.6[97]51.8 Â±1.5000000000Colony-formingÂ±3.149.9

Â±2.1[97[56.2 Â±3.953.0 Â±2.0Â¡98)85.0 Â±6.647.7 (4.4)6.1(1.2)11.6(2.2)77(1)77(1)121 Â±3.7[99]59.3 Â±3.151.3 Â±1.4Â¡98]83.9 Â±4.448.0 Â±4.2[95]63.0 Â±2.850.1

B2TExp.Conii.TreatmentNANA+Ca2*+RA+Ca2VRANANA+Ca2*+RA+Ca2VRANANA+Ca2*+RACholesterol Â±3.0[93[68.1 Â±2.851.3 Â±5.6(90]89.7 +4.048.7 (1.6)46 Â±6.1[97]70.8 Â±5.652.6 (0.6)["SJSulfate Â±4.3 J95JSpontaneous Â±1.5 " CE, cross-linked envelope; Exp., exponential phase; NA. no additions; (Dull.. 3 days after cell confluence; RA, retinoic acid. * Numbers in parentheses, fold increase. c Mean Â±SE. " Numbers in brackets, percentage of activity associated with the paniculate fraction.

notype as indicated by the production of cholesterol sulfate and the formation of cross-linked envelopes. This program of dif ferentiation is influenced by the concentration of calcium in the Ul medium and by the presence of retinoic acid. High calcium stimulates the expression of the differentiated phenotype as indicated by the increase in cholesterol sulfate accumulation CO and cross-linked envelope formation. In addition, the growth of the cells was inhibited in high calcium. These observations or UJ I coincide with, and add significantly to, earlier reports (6, 9) in (0 which we found that the nontumorigenic cells were larger and 111 _l more polygonal in shape and contained more tonofilaments O Q. and junctional complexes when grown in REM-1 medium o containing high (1 mM) calcium. In addition, the clonal growth of these cells was inhibited in high calcium (6,9). A stimulation

RE-2BT cells exhibited high levels of cholesterol sulfate and ACKNOWLEDGMENTS cholesterol sulfotransferase activity at all stages of growth. The technical assistance of Anthony Gallarti is most appreciated. These levels were independent of the presence of calcium or retinoic acid. These results suggest that the synthesis of choles terol sulfate in RE-B2T cells occurs in a constitutive manner REFERENCES and is no longer controlled by calcium or retinoids as is the 1. Day, N. E. Some aspects of the epidemiology of esophageal cancer. Cancer case for transglutaminase activity. The altered responses of RE- Res., 35: 3304-3307, 1975. B2 and B2T cells to calcium and retinoic acid, both of which 2. Schoenberg, B. S., Bailar, J. C., Ill, and Fraumeni, J. R., Jr. Certain mortality patterns of esophageal cancer in the United States, 1930-76, J. Nati. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1988 American Association for Cancer Research. Cholesterol Sulfate Accumulation in Tumorigenic and Nontumorigenic Rat Esophageal Epithelial Cells: Evidence for Defective Differentiation Control in Tumorigenic Cells

J. I. Rearick, G. D. Stoner, M. A. George, et al.

Cancer Res 1988;48:5289-5295.

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