Induction of Erythroid Differentiation in K562 Cells by Inhibitors of Inosine Monophosphate Dehydrogenase1

[CANCER RESEARCH 49. 5555-5560. October 15. 1989] Induction of Erythroid Differentiation in K562 Cells by Inhibitors of Inosine Monophosphate Dehydrogenase1

John Yu,2 Victor Lemas, Theodore Page, James D. Connor, and Alice L. Yu

Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037 fj. Y., V. L.], and Department of Pediatrics, University of California at San Diego, La Jolla, California 92093 [T. P., J. D. C., A. L. Y. ]

ABSTRACT by incubation with ribavirin were also analyzed. Preliminary reports of this investigation have been presented (15, 16). The effects of three inhibitors of inosine monophosphate (IMP) dehydrogenase on a human erythroleukemic cell line, K562, were studied. Following incubation with these inhibitors, K562 cells underwent differ MATERIALS AND METHODS entiation and accumulated hemoglobins. The induction of hemoglobin accumulation was dose dependent; maximum induction was observed at Cell Culture. Stock cultures of K562 (American Type Culture Col 100, 25, and 3 MM,respectively, for ribavirin, tiazofurin, and mycophen- lection, Rockville, MD) were grown in RPMI 1640 medium supple olic acid. The induction was associated with reduction of intracellular mented with 50 lU/ml of penicillin, 50 Mg/ml of streptomycin, and GTP content and was blocked by adding guanosine within 24 h after 15% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), as adding inducer. The effective dose for half-maximum induction by riba previously described (17). Cells were caused to differentiate by addition virin was 3 times less than that for 50% inhibition of K562 proliferation; of inducers to final concentrations as specified in the text. Ribavirin however, for tiazofurin and mycophenolic acid, it closely approximated (Viratek, Inc., Covina, CA), tiazofurin (gift from Dr. Roland Robins), the concentrations which suppressed cellular proliferation. Ribavirin was and mycophenolic acid (Sigma, St. Louis, MO) were diluted to 10 mg/ sequestered preferentially inside the K562 cells, and the induction by ml in RPMI 1640 medium and stored at 4Â°C.Experimental cultures ribavirin had a greater than 30-fold increase in hemoglobin. Studies with were grown for 3 to 4 days to densities less than 2x10'' cells/ml. Cell isoelectric focusing, globin chain analyses, and immunochemical assays numbers were counted with a hemocytometer. Cell viability was deter indicated that both Ay and Gy were detected and that the hemoglobin mined by trypan blue dye exclusion. The number of cells containing produced in the ribavirin-treated cells consisted of approximately 60% hemoglobin was assayed by benzidine staining (18). The clonogenic fetal hemoglobin and its acetylated equivalents. The adult-type a globin assay was performed using 0.3% agar in RPMI 1640 medium contain was found, while no ,t globin chains were demonstrated. Thus, accumu ing 15% fetal calf serum (19, 20). The amount of hemoglobin accu lation of fetal hemoglobin and production of a globin chain in ribavirin- mulation was assayed using a benzidine colorimetrie method (21). treated cells are different from the pattern of hemoglobins induced by GTP and ATP Assay. For the determination of intracellular GTP hernia. and ATP in K562 cells, samples were prepared as we described previ ously (22, 23). Briefly, K562 cells were incubated with ribavirin or tiazofurin. After a specified time of incubation, approximately 2 to 5 x INTRODUCTION 10" cells were removed, counted with a hemocytometer, and centrifuged for 5 min at 1000 rpm. The resulting cell pellets were immediately Ribavirin (1-ÃŸ-D-ribofuranosyl -1,2,4 -triazole -3 -carboxam- extracted with 100 ^1 of l M ice-cold HC1O4 at 4Â°C.After vortexing, ide), tiazofurin (2-/3-D-ribofuranosylthiazole-4-carboxamide), samples were spun at 4Â°Cat 10,000 rpm, and the supernatant was and mycophenolic acid (1-3) were shown to be inhibitors for collected. The supernatant was immediately neutralized with 4 M KOH, IMP dehydrogenase and growth of tumor cells. Their primary and the pH was carefully titrated to 7.4 with l M KOH. The final volume was recorded and samples were quickly frozen and stored at effect on purine metabolism appeared to be a marked depletion -20Â°C until final assay for ATP and GTP concentrations by high- of guanine nucleotide pools (3-6). In vivo studies have indicated pressure liquid chromatography as previously described (23). that ribavirin and other inhibitors of IMP dehydrogenase might Isoelectric Focusing. Approximately 1 x IO8cells of ribavirin-induced induce hematological changes. Administration of ribavirin in K562 were washed twice in phosphate-buffered saline (pH 7.4). Then high doses frequently results in a reversible anemia, and the one volume of packed cells was mixed with one volume of deionized effect is dose and time dependent (7). Tiazofurin was also found water and one-half volume of CC14. After mixing for 10 min and to cause myelosuppression in Phase I clinical trials (8). In centrifugation for another 10 min at 4Â°C,KCN was added to the addition, it was found that several inhibitors of IMP dehydrog supernatant collected to a final concentration of 100 ^g/ml, and the enase were potent inducers of myeloid maturation in the HL- sample was stored at â€”¿20Â°C.Isoelectricfocusing of the samples was 60 cell line (9-11). These inhibitors were also reported to performed using 1% agarose containing ampholites at pH 6 to 8 in LKB Multiphor II apparatus at a constant 15 watts and 4Â°Cfor 20 to promote the terminal maturation and inhibit the self-renewal of normal myeloid progenitors (12, 13). Several previous studies 30 min. The agarose gel was then fixed in 10% trichloroacetic acid and stained with benzidine for the presence of hemoglobins. explored the effects of ribavirin on mature erythrocytes (7); so Globin Chain Analysis. After isoelectric focusing of cell lysates, far, there have been few data on its effect on immature erythroid individual gel slices were excised from gel without prior fixation and precursor cells in vitro or in vivo. In this study we report the then stored in 20 u\ of ÃŸ-mercaptoethanolat 4Â°C.These samples were effects of ribavirin, tiazofurin, and mycophenolic acid on the denatured with 100^1 of buffer containing 6.7 Murea, 8.3% acetic acid, induction of erythroid differentiation of a human erythroleu 8.3% /3-mercaptoethanol, and 0.3 mg/ml of pyronin Y and boiled for 2 kemic cell line, K562 (14). The types of hemoglobin produced min prior to subsequent electrophoresis in 12% polyacrylamide gel containing 6 M urea, 2% Triton, and 5% acetic acid (24). Electropho Received 6/13/88; revised 11/4/88, 4/17/89, 7/7/89; accepted 7/20/89. resis was carried out in 5% acetic acid for 17 h at a constant current of The costs of publication of this article were defrayed in part by the payment 10 mA. Gels were stained using Coomassie blue. In some cases, the 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. globin chain composition of the induced K562 cells was further ana 1This work was supported in part by grants from the NIH (DK 37039 and CA lyzed by a combination of Western-blotting and immunochemical assay 40186). This is Publication 5408-BCR from the Research Institute of Scripps as follows. The urea-Triton-acid polyacrylamide gel prepared as de Clinic, La Jolla, CA. 2To whom requests for reprints should be addressed at Department of Basic scribed was electroblotted to nitrocellulose paper (25) and probed and Clinical Research. BCR3. Research Institute of Scripps Clinic, 10666 North sequentially with a 1:2000 dilution of rabbit anti-human hemoglobin F Torrey Pines Road. La Jolla. CA 92037. or hemoglobin A serum and biotin-conjugated anti-rabbit antibodies 5555

(Vector Laboratories, Burlingame, CA). The respective globin chains cells and/or a commitment to differentiation, clonogenic assay on the nitrocellulose paper were then analyzed with an avidin-biotin- (19, 20) was performed after various periods of preincubation glucose oxidase assay (26). with ribavirin (Table 1). The colonies derived from K.562 cells Quantitation of Ribavirin. Concentrations of ribavirin in cell extracts reached an average size of 9 to 16 cells per colony, after 3 days and culture media were quantitated with a competitive binding radio- of cultures. With prior exposure to ribavirin for 1 day, the size immunoassay as described previously (27). The antibody is specific for both ribavirin and its phosphorylated nucleotides and can detect con of colonies was significantly smaller (Table 1), although the centrations as low as 0.01 UM. The cell number was counted with a total number of colonies per plate remained similar to control hemocytomer. The intracellular concentrations were calculated on the cultures. However, when K562 cells were preincubated with basis that 1 ml of packed K562 cells contains 1.85 x 10s cells, which ribavirin for more than 2 days before plating, the total number translates to a volume of 5.4 x 10~9ml/cell. of colonies decreased significantly, and most of the colonies consisted of less than 4 cells/colony. These results indicated that exposure to ribavirin led to growth restriction of K562 RESULTS cells. As also shown in Fig. 1B was the response of K.562 cells after Effects of Ribavirin and Other IMP Dehydrogenase Inhibitors incubation with ribavirin in liquid culture for 3 days, when they on K562 Cells. Exposure of K562 cells to a wide range of became benzidine positive in a dose-dependent manner. Maxi concentrations of ribavirin resulted in a concentration-related mum induction was observed with ribavirin at 25 Mg/ml (=100 decrease in cellular proliferation (Fig. IA). The concentration /UM).The effective dose for half-maximum induction by ribavirin of ribavirin which caused 50% inhibition of cell proliferation was approximately 4 Mg/ml (â€”16UM). after 3-day incubation was approximately 10 Mg/ml or 40 MM The effects of other inhibitors of IMP dehydrogenases on (Fig. 1B). The viability of these ribavirin-treated cells remained K562 cells were also investigated. The addition of tiazofurin greater than 95% after 3 days of incubation (Fig. IB). In order and mycophenolic acid for 3 days induced K.562 cells to become to ascertain whether growth restriction by high concentrations benzidine positive in a dose-dependent manner (Fig. 2). Maxi of ribavirin reflects a cytotoxic effect on proliferation of K562 mum induction was observed at 25 ^M for tiazofurin and 3 pM for mycophenolic acid. The effective doses for half-maximum Ribavirincone. induction by tiazofurin and mycophenolic were approximately 2 and 0.3 pM, respectively. Both tiazofurin and mycophenolic acid restricted the growth of K562 cells in a dose-dependent manner; the concentrations for 50% inhibition of K562 prolif eration were 2 /Â¿Mfortiazofurin and 0.3 UMfor mycophenolic acid (Fig. 2). Sequestration of Ribavirin Inside K562 Cells. To determine the pharmacokinetics of ribavirin during the incubation of K562 cells, the intracellular concentrations of ribavirin were deter mined. After incubation of cells with 20 MM ribavirin, the intracellular concentration of ribavirin was approximately 1.5 /Â¿M;thisamounts to a 75-fold increase in concentration inside the cells over the ribavirin concentration in the medium (Fig. 3). The elevated intracellular concentration of ribavirin re mained relatively constant for up to 96-h incubation. When K562 cells were incubated with 100 pM ribavirin for 24 h, the 24 48 intracellular concentration increased to 11.5 MM,which reflects a 115-fold increase in intracellular concentration. After longer Incubation(lus) incubation with ribavirin, the intracellular concentration grad ually declined to approximately 50-fold of the extracellular concentration by 96 h. These experiments indicate that ribavirin was sequestered preferentially inside the K562 cells to at least 50-fold. Effect on Intracellular Concentrations of Purine Nucleotides. When K562 cells were incubated with ribavirin or tiazofurin, consistent alterations in purine metabolism were observed; the GTP level decreased within 24 h of incubation, and this decrease i persisted throughout the 4-day culture periods (Table 2). In the Â£ a treated cells, the decrease of intracellular GTP content became 3 more apparent when the intracellular GTP concentrations were normalized against the measured concentrations of intracellular 0 0.1 0.5 1.0 5.0 10.0 500 1000 ATP (Table 2). Ribavirin Concentration ,

Table 1 Clonogenic analysis ofK562 cells after preincubation with ribavirin K562 cells were preincubated in the RPMI 1640 medium in the presence or absence of 100 UMribavirin for a period of 5 h, l day, or 2 days. After preincubation, cells were washed extensively and plated, in triplicates, at 500 cells per ml of 0.3% agar in the culture medium without the addition of ribavirin. After 3 days of incubation, the number of colonies per plate and the cell number per colony were counted. no3-427.0 with(cells/colony)5-877.0 the following size distribution of colonies no. of TreatmentsFive colonies236.3(100) h of preincubation Control medium Â±3.0"(11.4)* Â±5.0 (32.6) 1.0 Â±3.0 (55.4) Â±2.3 (0.6) RibavirinOne 31.7 Â±4.7(14.6)28.0 103.6 Â±7.5(47.7)58.3 82.0 Â±12.0(37.7)154.5 08.8 217.3(100)249.6(100)

day of preincubation Control medium Â±13.3(11.2) Â±14.2 (23.4) Â±19.7(61.9) + 6.1 (3.5) RibavirinTwo 88.3 Â±5.8(36.3)26.7 130.3 +34.9(53.6)64.0 24.7 Â±3.0(10.1)127.0 014.6 243.3(100)232.3

days of preincubation Control medium Â±2.5(11.5) Â±4.6 (27.5) + 5.7(54.7) Â±8.3 (6.3) (100) RibavirinColony 42.0 Â±3.6 (61. 2). 26.0 Â±4.0 (37.8)9-1613 0.7 Â±1.2(1.0)>171.3 0Total 68.7(100) ' Mean Â±SD. *Numbers in parentheses, percentage of distribution of various sizes of colonies within each sample.

12.0

9.0

â€¢¿5-55 6.0 to u != Â«,

B 3.0 0 0.1 1.0 10 100 1000 Tiazofurin Concentration I

24 48 72 96 1000- Hours of Incubation Fig. 3. Accumulation of ribavirin in K562 cells upon incubation with ribavirin. K562 cells were incubated at I to 2 X 10s cells/ml with ribavirin at concentrations of 20 UM(A) and 100 JIM(â€¢).Atthe specific time interval of incubation, aliquots of approximately 2 to 5 x IO6cells were taken, and the intracellular concentration of ribavirin was determined as described in "Materials and Methods." The intracellular "ribavirin" includes phosphorylated ribavirins, because the antibody used detects both ribavirin and its phosphorylated nucleotides (27). The results were expressed as nmol/g of cell extract (assuming that 1 ml of packed cells yields l g of cell extract). The ribavirin concentrations in the culture medium to which 100 >i\i (â€¢)wereinitially added are also indicated in units of /-\i.

centrations of guanosine alone (Table 3). Furthermore, guanosine had no influence on erythroid differentiation in K562 cells if it was added 24 h postinduction or later. Neither does it affect 0 0.1 0.5 10 the induction of differentiation in K562 cells by hemin. Mycophenolic Acid (Â¡M\ Characterization of Hemoglobin Accumulation. In ribavirin- Fig. 2. Effect of tiazofurin and mycophenolic acid on proliferation and induc or hemin-induced K562 cells, the amount of hemoglobin accu tion of K562 cells. K562 cells were incubated, in triplicates, with the specified mulation and the extent of induction were found to be com concentrations of tiazofurin (A) and mycophenolic acid (B) for 3 days. Aliquots parable with a greater than 30-fold increase in hemoglobin were then analyzed for percentage of cells staining for hemoglobin with benzidine (â€¢)and for cell density (O). The variation of the data was within 10% among contents (Table 4). The lysates of these cells were analyzed by triplicate samples. Cell viability was more than 95% of the control. isoelectric focusing in order to identify the types of hemoglobins produced. The individual hemoglobins produced in the ribavi- decrease in the number of benzidine-positive cells was also rin-induced K562 cells were designated as hemoglobins 1 to 5 accompanied by a decline in the benzidine staining intensity of in Fig. 4A, Approximately 60% of hemoglobins, which accu these cells. At higher concentrations (50 and 100 AIM),there mulated in ribavirin-treated cells, was found to be hemoglobin was no significant difference between control and ribavirin- or F (Hbf) (Band 3 in Fig. 4A) and its acetylated form (Band 2) tiazofurin-treated samples. These results suggest that the inhib by comparison with hemoglobin standards (Fig. 4B). The he itory effect of guanosine on K562 induction by ribavirin or moglobin Band 2 in Fig. 4A was more acidic than hemoglobin tiazofurin became less pronounced, owing to a moderate in A (as demonstrated in the experiment of mixing samples with crease in hemoglobin-containing cells as induced by high con- standards) and had the same pi as the acetylated hemoglobin F 5557

Table 2 Perturbation of GTP and ATP contents Â¡nK562cells after exposure to ribavirin or tiazofurin The log-phase K562 cells were seeded at 1.8 x 10' cells/ml in the presence or AB absence of 100 /JM ribavirin or 25 >iM tiazofurin. After the specific time of incubation, aliquots of cells were removed and analyzed for GTP and ATP contents as described in "Materials and Methods." The assay method (22, 23) used for quanlitation of 1 nmol of various purine compounds gave an average of standard deviation of Â±0.04 nmol with an average coefficient of variation of 3.84%, as previously reported from the same laboratory (43). The average values of GTP/ATP ratios (Â±SD)for 4 days of incubation were 0.18 (Â±0.05).0.07 (Â± 0.02), and 0.04 (Â±0.01).respectively, for incubation with control medium, riba virin, and tiazofurin. IncubationGTP 18.070 Day 24.052.521.9228.945.652.20.140.060.04Day37.022.411.9028.427.146.30.250.090.04Day47.692.032.1041.525.744.10.190.080.05 cellsControlcontent, nmol/107 mediumRibavirinTiazofurinATP 4.641.971.5368.0

0_ ^^ 25 cellsControlcontent, nmol/107 4" mediumRibavirinTiazofurinGTP/ATP31.241.445.40.12

ratioControl mediumRibavirinTriazofurinDay 0.150.050.03Day

Table 3 Effect ofguanosine concentrations and time of addition on the accumulation of benzidine-staining cells (percentage} in K562 caused by various inducen K562 cells were incubated, in triplicates, with 100 nM ribavirin, 25 nM tiazofurin. 25 nM hemin, or without any inducer. To these samples, guanosine was added to a concentration specified in the table simultaneously with inducer or at a time of either 24 or 48 h postinduction. After 4-day incubation, aliquots of samples were taken and analyzed for the percentage of cells stained with Fig. 4. Identification of hemoglobin phenotypes in the cell extracts of ribavirin- benzidine reaction. and Ili-min iniliiivil K562 cells. K562 cells were induced with 100 f<\i ribavirin and 25 ^M of hemin. respectively. After 3 days incubation, cell lysates were Guanosine concentration (JIM) prepared, and isoelectric focusing was performed as described in "Materials and Methods." A. ribavirin-trealed cell extract; B, hemoglobin standards (Isolab, Inc.. 0 25 50 100 Akron, OH); and C, hemin-treated cell extract. The individual hemoglobin bands Guanosine added simultaneously in the ribavirin-treated cell extract were designated as hemoglobins / to 5. The with inducer positions for hemoglobin standards. HbA. HhF, HhS. and HbC. arc indicated. In No inducer 6 Â±1" 8 Â±1 18 Â±1 30 Â±2 hemin-treated cell extract (('), hemoglobins Portland, Bart's, and HbFarc labeled Ribavirin. 100/jM 85 Â±1 13 Â±3 18 Â±1 26 Â±2 as a. b. and c, respectively. Tiazofurin, 25 MM 88 Â±1 8Â±2 18 Â±1 29 Â±3 Hemin, 25 Â»JM 94 Â±I 95 Â±1 97 Â±1 94 Â±1 accumulated hemoglobins was the hemoglobin F, and the re Guanosine added 24 h postinduc maining 86% was hemoglobin Portland, Barts, and other uni tion Ribavirin. lOO^M 77 Â±4 78 Â±1 82 Â±1 89 Â±1 dentified hemoglobins (24, 28-30). Tiazofurin. 25 Â»IM 78 Â±1 83 Â±2 80 Â±2 81 Â±2 Analysis of Globin Chain Composition. The phenotypes of the hemoglobins that accumulated in the ribavirin-induced K562 Guanosine added 48 h postinduc tion cells were confirmed by globin chain analysis. The individual Ribavirin. 100 JIM 79 Â±2 80 Â±2 83 Â±2 hemoglobin bands in the isoelectric focusing gel (Fig. 5A) were Tiazofurin. 25 JIM 86 Â±1 85 Â±1 89 Â±1 87 Â±2 " Mean Â±SD of triplicate samples. excised. After elution and denaturation, the globin chains were analyzed in urea-Triton-acid polyacrylamide gel (24). As shown

Table 4 Hemoglobin contents in uninduced and induced A'562 cells in Fig. 5Ã„,the composition of globin chains for hemoglobin Band 1 in Fig. 5A is y globin chains (i.e., Gy and A7) and f K562 cells were incubated with hemin, ribavirin, or medium alone for 3 days and then cell lysates were prepared from these cells, as described below. Approx chain, confirming the presence of hemoglobin Portland (fc y2). imately 1 x IO7 cells were collected and washed twice in phosphate-buffered Hemoglobin Band 3, which is the major hemoglobin species saline. pH 7.4. and aliquots were taken for cell counts and benzidine staining accumulated in the lysates, is composed of the y globin chains (18). One volume of packed cells was then mixed with one volume of deionized water and 0.5 volume of CCI4 and centrifuged at 4'C. and the amount of along with a globin, confirming the accumulation of hemoglo hemoglobin in the supernatants was determined as described (21). bin F (ah 7:). Band 2 showed characteristic patterns of ace- content tylated 7 chains (Fig. 5B, sample 6), confirming that this per 10*cells(fig)0.133.75 InducersNone cells (%)7 hemoglobin is hemoglobin F,. Hemoglobin Band 4 is mainly composed of two embryonic globin chains, fand Â£,whileBand Hemin. 25 fiM 9492 Ribavirin. 100 , MHemoglobin 3.27Benzidine-staining 5 consists of Â«and Â£chains. They are thus shown to be the hemoglobins Gower-1 and -2, respectively. Therefore, the major species of globin chains which accumulated in cells after riba (HbF,) in the cord blood (picture not shown). As also indicated virin induction were the y globin chains and the Â«chains. In in Fig. 4/1, the remaining hemoglobins consisted of 21% he no instances was hemoglobin A (a? ft) detected in globin chain moglobin Portland (Band I), 8% Gower-1, and 11% Gower-2 analysis. These results were also confirmed by immunoblotting (Bands 4 and 5). On the other hand, similar experiments using of the gels in Fig. 5B with appropriate antibodies. Antibodies hemin-induced K562 cells (Fig. 4C) showed that only 14% of directed against hemoglobin F (Â«272)detected the presence of 5558 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1989 American Association for Cancer Research. EFFECTS OF IMP DEHYDROGENASE INHIBITORS ON K562 CELLS an inducer. The issue of "commitment" of K562 cells to "ter B minal differentiation" remains to be further investigated (e.g., 1 2 Ref. 20). However, these results also showed that ribavirin causes cytotoxic effects on K562 cells. The higher the concen tration and the longer the exposure to the inducer prior to plating, the lower was the cloning efficiency in the clonogenic assay. In the studies of HL-60 cells (37), it was proposed that Â£- the observed differentiation in HL-60 might result from an Ay- -2- N-acetyl adaptive cellular response to a toxic stress imposed by inducers. Gr- However, our data on ribavirin showed that the concentration HbC- for half-maximum induction by ribavirin was about 3 times less L5_ ~ than that for 50% growth inhibition. This would suggest that inhibition of cellular proliferation may not be a necessary prerequisite of cellular differentiation. It has been shown that ribavirin 5'-monophosphate is a potent inhibitor of IMP dehydrogenase (4), which is involved Fig. 5. Globin chain analysis for the hemoglobins produced after ribavirin in the de novo synthesis of GMP. In agreement with previous induction. A, isoelectric focusing. Cell lysates were prepared from ribavirin- reports (5), present studies showed that K562 cells exposed to induced K562, and isoelectric focusing was performed as described in Fig. 4. The ribavirin displayed significant decline in the level of GTP. individual hemoglobin bands were designated as hemoglobins / to 5. The isoelec tric focusing gels, a and b, which had the same cell lysates were shown to indicate Mycophenolic acid and tiazofurin also promote similar altera that hemoglobins .< and â€¢¿/couldbe separated during a shorter duration of tions in inlrace II11larlevels of GTP (3, 6). The fact that all these isoelectric focusing (Gel a) and that both have a similar pi after completion of isoelectric focusing (Gel b). The positions for hemoglobin standards are also inhibitors of IMP dehydrogenase induced cellular differentia indicated for comparisons. B, gel slices containing the individual hemoglobin tion suggests a possible role of GTP depletion in K562 differ bands (/ to 5 in A) were excised from the isoelectric focusing gel and labeled as entiation. It is of interest to note that the induced maturation Samples / to 5. Globin chain analysis was performed in urea-Triton-acid polyacrylamide gel (24), as described in "Materials and Methods." As standards for of myeloid leukemia cell line HL-60 is also accompanied by an various globin chains. HbF, and HbA derived from cord blood preparation were intracellular depletion of guanosine ribonucleotides (10, 11), used (Sample 6). which reflects a down-regulation of guanylate synthesis from IMP at the rate-limiting step mediated by IMP dehydrogenase 7 chains (in Samples 1, 2, and 3 of Fig. 5B), and both antibodies (9, 38, 39). The role of GTP depletion in ribavirin-induced specific to hemoglobin A (a: ÃŸ2)andhemoglobin F, respectively, differentiation in K562 is further strengthened by the findings demonstrated the production of Â«chains (in Samples 2, 3, and that guanosine, which could replenish the effect of GTP deple 5) in the ribavirin-treated K562. In addition, the presence of tion, was able to block the differentiation of K562 induced by hemoglobin F in the ribavirin- or tiazofurin-treated K.562 cells ribavirin or tiazofurin but not by an unrelated inducer, hemin. was independently confirmed by immunofluorescence studies Similar results were also reported in the blockage of induced of the cytospin preparations of the induced cells using mono maturation of HL-60 cells by guanosine (39). On the other clonal antibody specific for 7 chains (31) (picture not shown). hand, the accumulation of hemoglobins in K562 cells was preceded by a decrease in GTP concentration which occurred within 24 h of ribavirin treatment. Whether or not these find DISCUSSION ings suggest any association between depletion of GTP and "commitment" of K562 cells into differentiation (20) remains Many studies have suggested that alterations in the regulation of IMP dehydrogenase may modulate the capacity of cells to to be investigated. undergo proliferation (32, 33). Malignant transformation was Present studies indicate that ribavirin was sequestered pref found to associate with an increase in the activity of IMP erentially inside the K.562 cells. The results confirm previous dehydrogenase (34, 35) and various drugs which inhibit this reports that there is a selective accumulation of ribavirin in enzyme displayed significant antitumor activities (32,33). Stud mature human erythrocytes in vitro (40). They also agree with ies of fetal and regenerating hepatic tissues had demonstrated the in vivo observations that approximately 10 to 15% of the a direct relation between levels of this enzyme and proliferation total administered ribavirin was incorporated into mature red of these cells (36). In line with these findings, our current cells, while red cells accounted for only 3% of the total body studies indicated that the use of inhibitors of IMP dehydroge mass and that the ratio between plasma trough levels of ribavirin nase led to the suppression of K562 proliferation. On the other and red cell ribavirin was approximately 1 to 90 (40). Conceiv hand, it also resulted in the induction of hemoglobin produc ably, ribavirin is entrapped within the mature erythrocytes tion. The optimal concentrations for the induction of differen because it is phosphorylated upon entry into these cells. Riba tiation in K562 cells by tiazofurin and mycophenolic acid virin was shown to be rapidly phosphorylated in mature red closely approximated the concentrations that suppressed cel cells and mainly exists as the 5'-triphosphate (41). lular proliferation. The clonogenic assays also showed that Present studies using the same subclone of K562 cells showed these differentiating cells exhibited restricted potential for pro that hemoglobin F constitutes most of the accumulated hemo liferation (i.e., terminal differentiation). The longer the expo globin in ribavirin-treated cells, in contrast to the production sure to the inducer prior to plating, the smaller the colony size, of mainly embryonic hemoglobins in hemin-treated cells (28- because more cells would have already undergone terminal 31). In ribavirin-treated cells, Gy represents about 75% of the differentiation prior to plating. While most of the colonies in 7 globin chains, which is similar to the value found in normal the drug-pretreated K562 are benzidine positive in clonogenic fetuses and newborns (42). The induction of hemoglobin accu assay, not all the cells within the colonies are clearly stained mulation with ribavirin was also marked by the accumulation with benzidine. These results indicate that K.562 cells may not of a globin chains and the lack of ÃŸglobinproduction. The Â«/ fully be committed and may require the continuous presence of 7 ratio was estimated to be 0.77, and no hemoglobin Bart's was 5559

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1989 American Association for Cancer Research. EFFECTS OF IMP DEHYDROGENASE INHIBITORS ON K562 CELLS identified in these cells. In hemin-treated K562 cells, the a/y differentiation of the human erythroid cell line K562. Nature (Lond.), 309: 631-633, 1984. ratio was 0.11 in our studies and was reported to be in the 18. Orkin, S. H., Haros, P. 1., and Leder, P. Differentiation in erythroleukemia range of 0.08 and 0.30 by others (24, 28). Therefore, the pattern cells and their somatic hybrids. Proc. Nati. Acad. Sci. USA, 72: 98-102, of hemoglobin accumulation in ribavirin-treated cells was dif 1975. 19. Yu, J., and Smith, R. D. Sequential alterations in globin gene chromatin ferent from the a thalassemic imbalance observed in hemin- structure during erythroleukemia cell differentiation. J. Biol. Chem., 260: treated K562. These findings raise the possibility of using 3035-3040, 1986. 20. Rowley, P. T., Ohlsson-Wilhelm, B. M., and Farley. B. A. K562 human ribavirin to increase fetal hemoglobin production in patients erythroleukemia cells demonstrate commitment. Blood, 65: 862-868, 1985. with sickle cell disease and 0-thalassemia syndromes, if similar 21. Tsiftsoglou, A. S.. Gusella, J. F., Vollock, V., and Houseman. D. E. Inhibition by dexamethasone of commitment to erythroid differentiation in murine results could be demonstrated in vivo. erythroleukemia cells. Cancer Res.. 39: 3849-3855. 1979. 22. Page, T., Bakay, B., Nissinen, E., and Nyhan, W. L. Hypoxanthine-guanine phosphoribosyltransferase variants: correlation of clinical phenotype with ACKNOWLEDGMENTS enzyme activity. J. Inherited Metab. Dis., 4: 203-206, 1981. 23. Bakay, B.. Nissinen. E.. and Sweetman, L. Analysis of radioactive and We would like to thank Dr. T. Papayannopoulou. 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Accumulation of a heat shock-like protein during 706:497-505. 1980.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1989 American Association for Cancer Research. Induction of Erythroid Differentiation in K562 Cells by Inhibitors of Inosine Monophosphate Dehydrogenase

John Yu, Victor Lemas, Theodore Page, et al.

Cancer Res 1989;49:5555-5560.

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