[CANCER RESEARCH 41, 808-81 2, March 1981] 0008-5472/81/0041-OOOOS02.00 /V,/V-Dimethylformamide and Sodium Butyrate Modulation of the Activities of Purine-metabolizing Enzymes in Cultured Human Colon Carcinoma Cells1

Daniel L. Dexter,2 Gerald W. Crabtree, Johanna D. Stoeckler, Todd M. Bavarese,3 Lucy Y. Ghoda, Timothy L. Rogler-Brown, Robert E. Parks, Jr., and Paul Calabresi

Department of Medicine, Roger Williams General Hospital, Providence 02908 ¡D.L. D.. P. C.I, and Sections of Biochemical Pharmacology [G W. C.. J. D. S., T. M. S., L. Y. G , T. L. P-B., R. E. P.] and Medicine [D. L. D., P. C.], Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

ABSTRACT The polar solvents, DMSO" and DMF, and butyric acid or its sodium salt are among the chemicals used in studies with The specific activities of 11 purine-metabolizing enzymes Friend cells (9, 15). Furthermore, treatment of cultured rodent were assayed in three cultured human colon carcinoma cell tumor cells with these compounds has reduced tumorigenicity lines, HCT-15 and the A and D clones of the heterogeneous and clonogenicity in soft agar, decreased doubling times, and DLD-1 line. Enzyme activities were also determined for each of increased saturation densities. These changes have been re the cell lines cultivated in the presence of either of two com ported for DMSO-treated Friend cells (21 ), DMF-treated murine pounds known to induce differentiation in tumor cells: the polar rhabdomyosarcoma cells (5), and transformed Chinese ham solvent A/,A/-dimethylformamide and sodium butyrate. The ac ster cells exposed to butyric acid (14). tivities of three enzymes, purine nucleoside phosphorylase, 5'- There have been several recent reports on induction of better deoxy-5'-methylthioadenosine phosphorylase, and nucleoside differentiated or more benign phenotypes in cultured human diphosphokinase were not changed by exposure to dimethyl- cancer cells. DIVISO has effected cilia formation in human lung formamide or butyrate. Xanthine oxidase activity was not de tumor cells (30), and DMSO, DMF, and butyrate have induced tectable in any of the lines and was not inducible by either granulocytic differentiation in human leukemia cells (4). Kim et compound. Expression of seven other enzymes was modulated al. have shown that butyrate increases sialyltransferase activity more than three-fold in at least one line by one or both agents. in cultured human colon cancer cells (13), and butyrate has These enzymes include adenine and hypoxanthine-guanine also been shown to effect increased production of human phosphoribosyltransferases, adenosine kinase, adenosine de- chorionic gonadotropin in HeLa cells (16). Also, an increased aminase, guanine deaminase, and guanosine and adenosine synthesis of adult-type collagen in a human tumor line estab monophosphate kinases. Although both agents induce differ lished from a glioblastoma multiforme has been induced by entiation, striking differences in the patterns of enzyme modu treatment with hexamethylene bisacetamide (22). The effect of lations were observed. The effects were cell line dependent as DMF on cultured human colon carcinoma cells has been stud well as agent dependent. Guanine deaminase activity in HCT- ied in our laboratory. This polar solvent causes changes in the 15 cells increased six-fold upon exposure to butyrate, whereas growth properties of several colon cancer cell lines that are it decreased three-fold in clone D cells in response to dimethyl- consistent with those expected of a cell with a less malignant formamide. Adenosine deaminase activity in dimethylformam- phenotype. Thus, colon carcinoma cells grown in DMF-con- ide-treated clone A cells was decreased 11 -fold compared to taining medium have longer doubling time, lower saturation that determined for untreated cells. Therefore, some human density, complete loss of clonogenicity in soft agar, and mark colon tumor cells treated with dimethylformamide could be edly reduced tumorigenicity compared to the untreated tumor more sensitive to adenosine analogs such as formycin and 8- cells (6). Furthermore, DMF has induced several differentiated azaadenosine, which are good substrates for adenosine de features in these cells. An increase in cell surface carcinoem- aminase. These results support the concept that relatively bryonic antigen expression has been detected with exposure nontoxic differentiation-inducing compounds might potentiate to DMF, as well as decreased sensitivity to an antiserum the effects of antineoplastic drugs. directed against colonie mucoprotein antigen purified from human colon carcinoma tissue (11 ). An increased sensitivity to INTRODUCTION an antiserum directed against colonie mucoprotein antigen purified from normal human colonie epithelium was also meas Differentiation and the alteration of malignant to more benign ured in 2 of our lines following DMF treatment. DMF-treated phenotypes have been induced in a number of cultured tumor colon tumor cells also show a decreased expression of the H- cell types by chemicals. The most extensively characterized system for differentiation induction is the Friend virus-trans blood group determinant compared to untreated cells (11). formed murine erythroleukemia. The expression of the eryth- Therefore, cultured human colon carcinoma cells provide a model to demonstrate that DMF alters the malignant phenotype roid program in these cells has been effected by treatment with of these cells to a phenotype characteristic of a more benign many structurally unrelated compounds (2, 9, 15, 24, 27, 29). cell and that, concomitant with this alteration, there are differ ' This work was supported by Grants CA 07340, CA 13943. CA 20892, and entiation changes occurring in treated cells (7). The clinical CA 23225 from the USPHS and Grant CH-7T from the American Cancer Society. implications of these findings have been discussed in another 2 To whom requests for reprints should be addressed, at Department of Medicine, Roger Williams General Hospital. 825 Chalkstone Avenue, Providence, report from our laboratory (3). R. I. 02908. 3 Recipient of an Advanced Predoctoral Fellowship from the Pharmaceutical 4 The abbreviations used are: DMSO, dimethyl sulfoxide; DMF, W,W-dimethyl- Manufacturer's Association. formamide; ADA, adenosine deaminase (E.G. 3.5.4.4); MTA, 5'-deoxy-5'-meth- Received August 7, 1980; accepted November 17, 1980. ylthioadenosine.

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These studies have raised the question whether differentia Treatment of Cultures with Inducers. Cultures of human tion-inducing agents could be used in the treatment of cancer. colon carcinoma cells were grown in medium containing 0.8% Drugs which alter the malignant phenotype of tumor cells might DMF as described previously (6). This concentration of DMF act directly in vivo against the neoplastic cells to effect a has no toxic effects on these cells (6). Cells were also cultivated maturational therapy. Alternatively, these biological modifiers in 3 row sodium butyrate; this concentration of butyrate was might prime the target tumor cell and make it more vulnerable not toxic to any of the cell lines tested. In all induction studies, to conventional drug therapy. The experiments reported here cells were exposed to DMF or butyrate, these cultures were were carried out to determine whether DMF or butyrate had passaged in the presence of the compound at least twice, and any effect on the levels of purine-metabolizing enzymes in enzyme levels were determined on cells in their third to fifth human colon cancer cells. The reactions catalyzed by the passage in the drug. Enzyme levels in untreated cells were enzymes examined in this study are shown in Chart 1. The determined for cultures initiated at the same time as cultures basic biochemical data obtained from this study might be useful subsequently exposed to DMF or butyrate. We have deter for the development of rational protocols for testing combina mined that at least 2 passages of these cells in DMF-containing tions of purine analogs and differentiation-inducing drugs medium are necessary to achieve the full expression of the against human colon cancer cells in cell culture and in xeno- DMF-induced phenotype (5, 6, 11) and that 2 passages in graft model systems. Portions of this work have been presented butyrate medium are necessary to obtain the complete mor in preliminary form (8). phological changes caused by this fatty acid salt. Preparation of Homogenates. Cells were grown in 100-mm MATERIALS AND METHODS tissue culture dishes (Falcon Plastics, Oxnard, Calif.) until cultures were almost confluent; 15 plates were prepared tor Cell Lines. The establishment at the Roger Williams Cancer each cell type. Our medium formulation (Roswell Park Memorial Center of the human colon carcinoma cell lines HCT-15 and Institute Tissue Culture Medium 1640 supplemented with fetal DLD-1 and the isolation of clones A and D from the heteroge calf serum and antibiotics) and culture techniques have been neous DLD-1 line have been reported previously (6). Inocula described previously (6). The medium was decanted, and each tion of nude mice with clone A or clone D cells produces poorly plate was rinsed twice with 10 ml of 0.9% NaCI solution. Then, differentiated or moderately differentiated adenocarcinomas of 1 ml of a 0.1 M potassium phosphate buffer (pH 7.4) was added the colon, respectively. HCT-15 cells produce well to moder to the first dish, and the cells were removed by scraping with ately differentiated colonie adenocarcinomas when injected a rubber policeman. The slurry was transferred with a Pasteur into nude mice. Clones A and D differ in karyotype and mor pipet to the next dish, and the process was repeated until the phology, and the 3 lines differ in their cloning efficiencies in cells from the entire set of plates had been harvested. The cell soft agar and in their saturation densities. HCT-15, clone A, suspension was kept at 4°.Then, 0.5 ml of buffer was added and clone D cells all have doubling times of about 20 hr. to the first dish, and the sequential rinsing and scraping were Admettait

PRPP •PRA**«"TOAR RNA Gkitomirw

(a) HGPRT Hypoxantt*» (b) APRT (c) Adtnosint KinoM fpXonlîine-«-^-1«Uric

id) ADA *WUGuano« (*) PNP (f) AMP Kinase (g) 6MP Kinase AcidGUMl(a)kin*(If* n« (h) NO P Kinase (I) AMP DtaminoM (j) Xanthiiw OxidcM (k) GuanoM REACTIONS OF PURINE METABOLISM (I) MTA Phosphorylas« Chart 1. Reactions of purine metabolism catalyzed by enzymes whose activities were examined in this study. PRPP. S-phosphoribosyl-1-pyrophosphate; PRA. ribosylamine phosphate; FGAR, phosphoribosyl formylglycineamide; FGAM, phosphoribosyl formylglycineamidine; XMP, xanthosine-5'-monophosphate; AMPS. adenylosuccinate; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; APRT. adenine phosphoribosyltransferase; PWP, purine nucleoside phosphorylase; NDP kinase, nucleoside diphosphokinase.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. D. L Dexter et al. repeated for all dishes. This wash volume was then added to methods identical to those used by Agarwal ef al. (1 ). the initial slurry, the entire volume was transferred to a 15-ml A modification of the coupled spectrophotometric assay de homogenizer, and the cells were broken with 15 strokes. The scribed by Pegg and Williams-Ashman (20) was used to deter homogenate was centrifuged for 1 hr at 100,000 x g in a mine the activity of MTA phosphorylase. Assays were carried Beckman L2-65 ultracentrifuge at 5°. The supernatant fluid out at 37°.Each cuvet contained, in a final volume of 1 ml, 20 was removed with a Pasteur pipet and was used immediately mw potassium phosphate buffer (pH 7.4), 0.8 unit of xanthine in the enzyme assays. The pellet was also used for determi oxidase, 500 /tw MTA, and appropriate amounts of the cell nations of adenosine kinase (EC 2.7.1.20) activity. extracts (up to 400 /ig protein). Since the presence of sulfhydryl Enzyme Assays. All enzyme assays were performed at room reagents is required for maximal MTA phosphorylase activity temperature unless indicated otherwise. A modification of the (32), cell extracts were pretreated with dithiothreitol (1 mw final radiochemical method of Kelley ef al. (12) was used for the concentration) for at least 2 hr at 4° before assays were determination of adenine phosphoribosyltransferase (EC performed. 2.4.2.7) and hypoxanthine-guanine phosphoribosyltransferase In almost all cases, enzyme activities were assayed on the (EC 2.4.2.8) activities. Reaction mixtures (500 /il) contained same day as the extracts were prepared. In a few instances, 0.1 M Tris-acétate buffer (pH 7.4), 5 mw MgCI2, 1 mw 5- extracts were stored at —20°for 24 to 48 hr before assays phosphoribosyl-1-pyrophosphate, extract (25 or 50 /il), and were performed. In the latter cases, the activities of enzymes either 0.1 mM[8-14C]adenine (specific activity, 11 .OmCi/mmol) examined after such storage were found to be negligibly or 0.1 HIM [8-14C]hypoxanthine (specific activity, 9.9 mCi/ changed from those seen with fresh extracts. For the purposes mmol). Aliquots (100 /¿I)ofthe reaction mixtures were removed of this study, enzyme activities are presented as the nmol of and added to 20 /il of 4 M formic acid to terminate the reactions substrate converted to product per min per mg protein. Protein at appropriate times after initiation of the reaction by addition concentrations were measured by the method of Lowry ef al. of cell extract. Nucleotide products were separated from the (17). precursor base using thin-layer chromatography by spotting Chemicals. The following chemicals were obtained from 10-/il aliquots of the formic acid extracts on microcrystalline commercial sources: 5-phosphoribosyl-l -pyrophosphate, ad cellulose thin-layer chromatography sheets (J. T. Baker Chem enine, hypoxanthine, MTA, and milk xanthine oxidase (Grade ical Co., Phillipsburg, N. J.) which had been spotted previously III; Sigma Chemical Co., St. Louis, Mo.); adenosine, AMP, ADP, with the appropriate nonradioactive base and nucleotides as ATP, and IMP (P-L Biochemicals, Inc., Milwaukee, Wise.); and carriers. Plates were developed in 5% Na2HPO4 for about 1 hr. dithiothreitol (Calbiochem-Behring, La Jolla, Calif.). Samples of Following development, areas corresponding to the base and 2'-deoxycoformycin [(fî)-3-(2-deoxy-/J-D-erythropentofurano- nucleotides were visualized by UV (254 nm), cut out, and syl)-3,6,7,8-tetrahydroimidazo[5,4-d][1,3]diazepin-8-ol] were counted in a toluene-based liquid scintillation system [15 g provided by Dr. H. W. Dion of Parke-Davis, Detroit, Mich., and Omnifluor (New England Nuclear, Boston, Mass.) in 1 gallon by Dr. John Douros of the National Cancer Institute, Bethesda, scintillation-grade toluene]. Nucleotide formation was linear Md. The following radiochemicals were purchased from com with respect to both time and protein concentration under these mercial sources: [8-14C]adenine and [8-14C]hypoxanthine conditions. (Schwarz/Mann, Orangeburg, N. Y.); [8-'"C]adenosine (New A radiochemical assay was also used to determine adenosine England Nuclear, Boston, Mass.). kinase activity. The reaction mixture (500 /il) contained: 100 mw Tris-HCI buffer (pH 7.0), 50 /IM [8-14C]adenosine (specific RESULTS activity, 10 mCi/mmol), 0.5 mw MgCI2, 1 HIMATP, 10 PIM NaF, 5 HIM dithiothreitol, 2 /¿M2'-deoxycoformycin, and up to 250 Effects of Compounds on Cell Morphology and Growth. /tl of extract. The extract was preincubated for 10 min in the Growth of colon cancer cells in DMF resulted in the drug effects presence of the ADA inhibitor 2'-deoxycoformycin before the typically observed in our laboratory (6). The cells grew more reaction was started by the addition of labeled adenosine. At slowly, became more angular in appearance, and did not pack appropriate times after initiation of the reaction, 50-/tl aliquots as tightly in colonies as did untreated cells. Cultures exposed of the reaction mixture were withdrawn and added to 10 /il of to DMF and passaged in DMF were routinely 95% viable by formic acid at 4°to terminate the reaction. After centrifugation trypan blue dye exclusion and replated as well as untreated for 10 min at 15,000 x g, 10-/il samples of the supernatants cells at the expansion plating densities used in these studies. were spotted on thin-layer chromatography sheets (see above) Sodium butyrate was added to cultures in varying concentra spotted previously with unlabeled adenosine and AMP as car tions, and 3 mw was found to be optimal in all lines studied for riers. Sheets were developed in 95% ethanol:1 M ammonium effecting growth inhibition and morphological changes without acetate, pH 7.5 (7:3). Areas corresponding to adenosine and concomitant loss in cell viability or plating efficiency at the adenine nucleotides were visualized by UV (254 nm), and their transfer densities used in these experiments. As with DMF- constituent radioactivity was determined by liquid scintillation treated cultures,butyrate-treated cells were always 95% viable. counting (see above). However, the morphological changes observed following ex ADA (25), purine nucleoside phosphorylase (EC 2.4.2.1) posure of cultures to butyrate were even more pronounced (28), and guanine deaminase (EC 3.5.4.3) (10) activities were than those seen in DMF-treated cells. The increased angularity measured by published procedures. Xanthine oxidase assays and inability to pack closely in colonies were especially pro were performed essentially as described by Massey ef al. (19), nounced for clone A cells. except that reactions were carried out at 30°.The activities of Effects of Compounds on Enzyme Activities. The specific AMP kinase (EC 2.7.4.3), GMP kinase (EC 2.7.4.8), and nu activities of 11 purine-metabolizing enzymes (names given in cleoside diphosphokinase (EC 2.7.4.6) were measured by legend to Chart 2) were assayed in 3 human colon carcinoma

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Modulation of Purine Enzymes in Colon Cancer Cells cell lines established at the Roger Williams Cancer Center. Enzyme levels were also determined for each of the cell lines Sa cultivated in the presence of DMF or sodium butyrate. For a rFOLDIa I~S change to be considered significant in this study, the activity of 5 8fe ¡ I H *•2 an enzyme had to increase or decrease by a factor of at least CHANGEFROM 3-fold following exposure of a cell to DMF or butyrate. The 3CONTROL ÃŒilli-"I 1 \11 ì activities of 3 enzymes, purine nucleoside phosphorylase, MTA 1S32•~ *u! -y ÕJl J, .i • phosphorylase, and nucleoside diphosphokinase, were not treotM? •OMF- changed in any of the lines by either DMF or butyrate exposure. Butyrott-tr«ot«dia1V1•" D A fourth enzyme, xanthine oxidase, was not detected in any of the lines, and neither DMF nor butyrate treatment induced Chart 4. Modulation of activities of enzymes of purine metabolism in cultured activity. The activities of 7 other enzymes were modulated HCT-15 human colon adenocarcinoma cells after treatment with DMF or butyrate. more than 3-fold in at least one of the lines by one or both For details, see the legend to Chart 2. compounds; these results are presented graphically in Charts 2 to 4. levels were affected by both compounds; there was a differ Adenine phosphoribosyltransferase, hypoxanthine-guanine ence among lines for the sensitivity to modulation of guanine phosphoribosyltransferase, adenosine kinase, and GMP kinase deaminase and ADA, however. Clones A and D showed de activities were significantly decreased only in butyrate-treated creased ADA levels with DMF treatment; butyrate had no real clone D cells. DMF had no significant effect on the levels of effect on ADA activities in these clones. The 11-fold decrease these 4 enzymes. AMP kinase activity was modulated only in of ADA activity measured when clone A cells were exposed to clone D cells and only by DMF. Guanine deaminase and ADA DMF was the biggest shift in the activity of an enzyme seen in this study. The converse was true with HCT-15 cells; butyrate induced an increase in ADA activity, whereas DMF had no effect. With HCT-15 cells, there was almost a 6-fold butyrate- effected increase of guanine deaminase activity; DMF had no E feaeS§ilïIlilITllTiiti^^^llTnll«'--_-J effect on guanine deaminase in these cells. The guanine de aminase level in clone D cells was not affected by butyrate but --• - • | was decreased significantly by DMF treatment. FOLDCHANGEFROMCONTROLi313S79r

DMF-tr«t«dO DISCUSSION 8utyrat«-treot«di^_ The selective potentiation of antineoplastic agents by rela tively nontoxic differentiation-inducing compounds constitutes a somewhat novel approach for improvement of single and multimodality treatments. This study shows that the activities of several purine-metabolizing enzymes in cultured human Chart 2. Modulation of activities of enzymes of purine metabolism in cultured colon cancer cells can be modulated by DMF or butyrate. DLD-1 clone A human colon adenocarcinoma cells after treatment with DMF or These results suggest that the efficacies of purine analogs sodium butyrate. Changes are expressed as the fold change of the mean of treated values from that of control values (taken as 1.0, horizontal line) after against cancer cells might be increased by developing proto treatment with DMF (•)or butyrate (D). Control (untreated) enzyme activities cols that use a combination of analog plus inducer. For exam (mean ±S.D.) are presented in parentheses as nmol of substrate converted to ple, the use of agents like DMF to effect a decrease in the level product per min per mg protein. Columns above the line represent increased enzyme activities, those below the line show decreased activities. Each column of activity of a drug-metabolizing enzyme in tumor cells could represents the average of at least 2 determinations. For details of the enzyme prevent the inactivation of the conventional drug. The DMF- assays, see "Materials and Methods." APRT, adenine phosphoribosyltransfer ase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; ARK, adenosine induced inhibition of ADA activity in clone A and clone D cells kinase; PNP, purine nucleoside phosphorylase; G'ase, guanine deaminase; MTA- reported here illustrates this concept. Thus, such cells treated P, MTA-phosphorylase; AMPK, AMP kinase; NDPK, nucleoside diphosphokinase; with DMF could be more sensitive to drugs such as formycin GMPK, GMP kinase. and 8-azaadenosine, which are good substrates for ADA. It is important, of course, that the analog potentiation by inducer be selective for tumor versus normal cells in order to

Sì = -^ Ô055 — avoid a concomitant increase in host toxicity. Since the effects *' +i 0 ! l s i of DMF and butyrate are reversible, it is not presently known whether these agents can be used to treat xenograft tumors in FOLD CHANGE nude mice effectively, i.e., at doses where antitumor response FROM occurs with little or no host toxicity. Also, since the 3 lines CONTROL responded in varied ways to each of the 2 compounds, it is unlikely that one inducer would be effective against all human •DMF-treoted D Butyrate ~tr«ot«d colon tumors. Experiments are currently in progress in which nude mice bearing human colon tumor xenografts are treated with DMF or butyrate to determine whether an antitumor effect Chart 3. Modulation of activities of enzymes of purine metabolism in cultured DLD-1 clone D human colon adenocarcinoma cells after treatment with DMF or can be achieved. sodium butyrate. See the legend to Chart 2 for details. The experiments reported here suggest that several factors

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. D. L. Dexter et al. are involved in the modulation of purine enzyme activities. As in cultured human colon carcinoma cells. Cancer Res., 39. 1020-1025. 1979. seen in Charts 2 to 4, the modulation of enzymes catalyzing 7. Dexter. D. L., and Hager, J. C. Maturation-induction of tumor cells using a metabolic changes in purines is complex. Where changes human colon carcinoma model. Cancer (Phila.). 45: 1178-1184. 1980. occur with exposure to an inducer, they can be agent depend 8. Dexter, D. L., Parks, R. E., Jr., and Calabresi, P. Sodium butyrate and N,N- Dimethylformamide modulation of the activities of purine metabolizing en ent and also cell line dependent. It remains to be determined zymes in cultured human colon cancer cells. Proc. Am. Assoc. Cancer Res.. whether the modulation by an inducer of the level of a given 21: 30, 1980. enzyme can be achieved generally with most human colon 9. Friend, C. W.. Scher. W.. Holland. J. G., and Sato. T. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid carcinomas or whether it is an individual tumor-dependent differentiation by dimethylsulfoxide. Proc. Nati. Acad. Sei. U. S. A., 68. 378- property. Work is in progress to analyze other human colon 382, 1971. tumor cell lines to see whether the purine-metabolizing en 10. Giusti, G. Guanase. In: H. V. Bergmeyer (éd.),Methods of Enzymatic Analysis, Vol. 2, pp. 1086-1091. New York: Academic Press, Inc., 1974. zymes can be separated into categories including (a) modula 11. Hager, J. C.. Gold, D. V., Barbosa, J. A., Fligiel, Z., Miller. F., and Dexter. tion resistant, (b) modulation sensitive but agent specific, and D. L. N.W-Dimethylformamide-induced modulation of organ- and tumor-as sociated markers in cultured human colon carcinoma cells. J. Nati. Cancer (c) modulation sensitive for both inducers. The establishment Inst., 64: 439-446, 1980. of such a classification system would help greatly in the rational 12. Kelley, W. N., Rosenbloom, F. M.. Henderson. J. F., and Seegmiller, J. E. A design of protocols combining an inducer and a purine analog, specific enzyme defect in gout associated with the overproduction of uric acid. Proc. Nati. Acad. Sei. U. S. A., 57: 1735-1739, 1967. because both specificity with respect to inducer and the direc 13. Kim, Y. S., Tsao, D., Siddiqui, B.. Whitehead, J. S., Arnstein, P., Bennett, J., tion of the modulation will be critical determinants of potentia- and Hicks, J. Effects of sodium butyrate and dimethylsulfoxide on biochem tion in combination therapy. ical properties of human colon cancer cells. Cancer (Phila.). 45: 1185- 1192, 1980. Our results show that for any given cell type the enzyme 14. Leavitt, J., Barrett, J. C.. Crawford, B. D., and Ts'o. P. O. P. Butyric acid patterns associated with DMF and butyrate treatment are dif suppression of the in vitro neoplastic state of Syrian hamster cells. Nature (Lond.), 271: 262-265, 1978. ferent. This suggests that the 2 compounds may act via differ 15. Leder, A., and Leder, P. Butyric acid, a potent inducer of erythroid differ ent mechanisms, in agreement with reports from other labora entiation in cultured erythroleukemic cells. Cell, 5. 319-322, 1975. tories. Studies by Leder and Leder (15), Rovera and Bonaiuto 16. Leiblich, J. M., Weintraub, B. D., Rosen, S. W., Ghosh, N. K., And Cox, R. P. Secretion of hCG-o subunit and hCG by HeLa strains. Nature (Lond.), (26), and Rovera and Surrey (27) have shown that the polar 265: 746. 1977. solvent DMSO, quite similar to DMF in its properties, probably 17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein induces erythroid differentiation in Friend cells by a mechanism measurement with the Folin phenol reagent. J. Biol. Chem., 793. 265-275, 1951. different from that operating in butyrate induction of these 18. Lyman, G. H., Priesler, H. D., and Papahadjopoulos. D. Membrane action of cells. Moreover, it has been reported that the extent of mor DMSO and other chemical inducers of Friend leukemic cell differentiation. Nature (Lond.), 262: 360-363. 1976. phological differentiation observed when human promyelocytic 19. Massey, V., Brumby, P. E., and Komai, H. Studies of milk xanthine oxidase: leukemia cells are treated with buryric acid is more limited than some spectral and kinetic properties. J. Biol. Chem.. 244: 1682-1691. that observed with cells exposed to DMSO or DMF (4). The 1969. 20. Pegg, A. E., and Williams-Ashman, H. G. Phosphate-stimulated breakdown hypothesis that the plasma membrane plays an important role of 5'-methylthioadenosine by rat ventral prostate. Biochem. J., 115: 241- in polar solvent treatment of cells is supported by the finding 247, 1969. that DMSO decreases membrane fluidity (18). There is evi 21. Priesler, H. D.. Lutton. J. D., Giladi, M.. Goldstein, K., and Zanjani, E. D. Loss of clonogenicity in agar by differentiating erythroleukemic cells. Life dence that a primary effect of butyrate on cells is the inhibition Sci., 76: 1241-1252, 1975. of the deacetylation of nuclear proteins (23, 31). Therefore, 22. Rabson, A. S., Stern, R., Tralka, T. S.. Costa. J.. and Wilczek, J. Hexame- butyrate may cause differentiation changes in tumor cells thylene bisacetamide induces morphologic changes and increased synthesis of procollagen in cell line from glioblastoma multiforme. Proc. Nati. Acad. through a mechanism involving nuclear rather than membrane Sei. U. S. A., 74: 5060-5064, 1977. events. Our spectrum of human colon cancer cell lines and 23. Reeves, R., and Cserjesi, P. Sodium butyrate induces new gene expression purine-metabolizing enzymes provides a tool for the study of in Friend erythroleukemic cells. J. Biol. Chem., 254: 4283-4290, 1979. 24. Reuben, R. C., Wife, R. L., Breslow. R., Rifkind, R. A., and Marks, P. A. A mechanisms operating in the alteration of malignant pheno- new group of potent inducers of differentiation in murine erythroleukemia types by 2 distinct inducers: butyrate and the polar solvent, cells. Proc. Nati. Acad. Sei. U. S. A., 73: 862-866, 1976. 25. Rogler-Brown, T., Agarwal, R. P., and Parks, R. E., Jr. Tight-binding inhibi DMF. tors. VI. Interactions of deoxycoformycin and adenosine deaminase in intact human erythrocytes and Sarcoma 180 cells. Biochem. Pharmacol., 27: 2289-2296, 1978. REFERENCES 26. Rovera, G., and Bonaiuto, J. The phenotypes of variant clones of Friend mouse erythroleukemic cells resistant to dimethyl sulfoxide. Cancer Res., 36: 4057-4061, 1976. 1. Agarwal, R. P., Scholar. E. M.. Agarwal. K. C.. and Parks. R. E., Jr. 27. Rovera, G., and Surrey, S. Use of hypersensitive variant clones of Friend Identification and isolation on a large scale of guanylate kinase from human cells in analysis of mode of action of inducers. Cancer Res., 38: 3737- erythrocytes: effects of monophosphate nucleotides of purine analogs. 3744, 1978. Biochem. Pharmacol., 20: 1341-1354. 1971. 28. Stoeckler. J. D., Agarwal, R. P., and Parks, R. E., Jr. Purine nucleoside 2. Bernstein. A., Hunt. D. M., Crichley, V.. and Mak, T. W. Induction by ouabain phosphorylase from human erythrocytes. Methods Enzymol., 51: 530-538, of hemoglobin synthesis in cultured Friend erythroleukemic cells. Cell. 9: 1978. 375-381. 1976. 29. Terada, M., Epner, E., Nodel, V., Salmon, J., Fibach, E., Rifkind, R. A., and 3. Calabresi, P., Dexter. D. L.. and Heppner, G. H. Clinical and pharmacological Marks. P. A. Induction of murine erythroleukemia differentiation by actino- implications of cancer cell differentiation and heterogeneity. Biochem. Phar mycin D. Proc. Nati. Acad. Sei. U. S. A., 79 2795-2799, 1978. macol., 28: 1933-1941, 1979. 30. Tralka, T. S., and Rabson, A. S. Cilia formation in cultures of human lung 4. Collins, S. J., Ruscelli, F. W., Gallagher. R. E., and Gallo, R. C. Terminal cancer cells treated with dimethyl sulfoxide. J. Nati. Cancer Inst., 57:1383- differentiation of human promyelocytic leukemia cells induced by dimethyl 1388. 1976. sulfoxide and other polar compounds. Proc. Nati. Acad. Sei. U. S. A., 75. 31. Vidali, G., Boffa, L. C., Bradbury, E. M., and Allfrey. V. G. Butyrate suppres 2458-2462, 1978. sion of histone deacetylation leads to accumulation of multiacetylated forms 5. Dexter, D. L. W.fV-Dimethylformamide-lnduced morphological differentiation of histones H3 and H4 and increased DNase I sensitivity of the associated and reduction of tumorigenicity in cultured mouse rhabdomyosarcoma cells. DNA sequences. Proc. Nati. Acad. Sei. U. S. A., 75: 2239-2243, 1978. Cancer Res., 37: 3136-3140. 1977. 32. Zappia, V., Oliva. A., Cacciapuoti, G., Galletti, P., Mignucci, G.. and Carteni- 6. Dexter, D. L., Barbosa, J. A., and Calabresi, P. N.W-Dimethylformamide- Farina, M. Substrate specificity of 5'-methylthioadenosine phosphorylase induced alteration of cell culture characteristics and loss of tumorigenicity from human placenta. Biochem. J.. 775: 1043-1050. 1978.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. N,N-Dimethylformamide and Sodium Butyrate Modulation of the Activities of Purine-metabolizing Enzymes in Cultured Human Colon Carcinoma Cells

Daniel L. Dexter, Gerald W. Crabtree, Johanna D. Stoeckler, et al.

Cancer Res 1981;41:808-812.

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