[CANCER RESEARCH 52. 949-954. February 15. 1992] Changes in , -associated , and Intermediate Filaments during the Differentiation of HL-60 Leukemia Cells Mun-Fai Leung, John A. Sokoloski, and Alan C. Sartorelli1

Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT as , have been described in both differentiated granu locytic and monocytic leukemia cells (8-10), and an alteration The is composed mainly of microtubules (MT), microfil in the distribution of vimentin has also been observed in leu aments, and intermediate filaments (IF) that form a structural network kemia cells induced to undergo monocytic differentiation (11, which connects cellular membranes, cytoplasmic , and the nucleus. Since the cytoskeleton may be involved in modulating signal 12). Thus, although a variety of changes have been documented transduction and in the morphological and structural changes that occur during the initiation of the differentiation of leukemic cells by during cellular proliferation and differentiation, cytoskeletal changes a variety of inducing agents (9), a detailed analysis of cytoskel were measured by immunofluorescence microscopy and fluorescence- etal changes during the maturation process has not been carried activated sorter analysis during the differentiation of HL-60 leukemia out. cells induced by retinoic acid (RA). Differentiated HL-60 cells exhibited Since the cytoskeleton is an important scaffold upon which increased staining intensity and altered organization of MT and IF, as the processes of growth and differentiation are accomplished, visualized by immunofluorescence microscopy with anti- monoclo a characterization of the organization, the dynamic exchange nal and anti-vimentin antibody, respectively. A new procedure between soluble and polymerized pools of cytoskeletal proteins, was developed and used to measure the content of the cytoskeletal components of HL-60 cells during the process of maturation. HL-60 cells and the interaction between different cytoskeleton proteins that were fixed with formaldehyde in an MT-stabilizing buffer, permeabilized occur during maturation would appear to be important to our using L-lysophosphatidylcholine, stained for immunofluorescent measure understanding of the transition to a more differentiated phe- ment with specific for particular cytoskeletal components, and notype. In this report, we describe detailed changes in cyto analyzed by flow cytometry. Terminally differentiated cells produced by skeletal components, including MT, MAPs, MAP2 and tau, exposure to RA contained larger amounts of MT and the IF vimentin. and the IF, vimentin, of HL-60 leukemia cells induced to During the course of the maturation process, a transient increase in the differentiate along the granulocytic pathway by RA. Cytoskel amounts of the microtubule-associated proteins, (MAPs) MAP2 and tau, etal components were visualized by immunofluorescence mi occurred. An RA-supersensitive clone, designated HL-60/S4, and an RA- croscopy, and the content of the cytoskeletal components was resistant clone, designated HL-60/R3, were developed by mutagenization quantified by a newly developed method of immunofluorescence and selection. Use of these clones supported the concept that the observed staining followed by flow cytometric analysis. An RA-supersen changes in MT, MAPs, and vimentin were associated with the differen sitive clone called HL-60/S4 and an RA-resistant clone desig tiation process rather than being due to other effects produced by the nated HL-60/R3 were developed by mutagenization and selec retinoid. Thus, the findings suggest that changes in MT, MAPs, and IF are important to the terminal maturation of leukemia cells. tion, and these clones were used as tools to link changes in cytoskeletal components to the differentiation process rather than to other effects of RA. INTRODUCTION

The functions of the cytoskeleton include the maintenance of MATERIALS AND METHODS morphology, cellular motility, and phagocytosis; the formation and Treatments. HL-60 human promyelocytic leukemia of the mitotic spindle during cell division; and the transport or cells were provided by Dr. Robert C. Gallo of the National Cancer localization of cytoplasmic organelles (1). To accomplish these Institute, Bethesda, MD. HL-60 cells were routinely passaged in RPMI functions, the cytoskeleton forms a structural network of tu 1640 medium with 10% fetal bovine serum. Stock cell cultures were bules and fibrils, composed of three major elements: MT,2 passaged twice weekly and maintained in a humidified 37°Catmosphere , and IF, which connect the plasma membrane containing 5% CO2 in air. Cell stocks were screened routinely for to cytoplasmic organelles and the nucleus (2). An increase in Mycoplasma by the probe method (Gen-Probe, Inc., San Diego, MT has been reported in HL-60 leukemia cells induced to CA). differentiate along the granulocytic pathway (3), and an increase Clones HL-60/S4 and HL-60/R3 were developed by incubating HL- in the cellular levels of and actin-binding proteins has 60 cells with 10 fiM A'-methyl-W-nitro-W-nitrosoguanidine (Aldrich been observed in both murine (4, 5) and human (6, 7) differen Chemical Co., Inc., Milwaukee, WI) for 2 h, washing with fresh growth medium 3 times, and incubating cells in fresh growth medium for 3 tiated myeloid leukemia cells. Furthermore, changes in the days. Single-cell suspensions of the mutagenized cells were prepared expression of several cytoskeleton-associated proteins, as well using a Becton Dickinson fluorescence-activated cell sorter (Mountain View, CA). Nonviable cells were stained with 1 fig/ml of propidium Received 9/3/91; accepted 12/2/91. The costs of publication of this article were defrayed in part by the payment iodide (Sigma Chemical Co., St. Louis, MO) in PBS and were elimi of page charges. This article must therefore be hereby marked advertisement in nated during cell sorting. RA-sensitive and -resistant clones were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed, at Department of screened using a cytochrome c reduction assay described by Catino and Miceli (13) and NBT reduction as described below after clones were Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510. incubated with 10 TJMor 1 /¿MRAfor 4 days. 2The abbreviations used are: MT, microtubules; IF, intermediate filaments; In all experiments, 1 x 10s exponentially growing cells were seeded/ RA, retinoic acid; MAPs, microtubule-associated proteins; PBS, phosphate- ml of fresh growth medium. Cells were incubated with different con buffered saline; NBT, nitroblue tetrazolium; FITC, fluorescein isothiocyanate; centrations of all-fra/ts-RA (Sigma) for 1-5 days. A stock solution of 1 PM2G buffer, 0.1 M l ,4-piperazinediethanesulfonic acid, pH 6.9-1 niM MgSCV 2 m\i ethyleneglycol bis(/3-aminoethyl ether)-/v°,Ar,^V"^V'-tetraaceticacid-2 M HIMRA was prepared in 100% ethanol and stored at -20"C. glycerol. Measurement of HL-60 Cell Proliferation and Differentiation. Cell 949

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. CVTOSKELETON CHANGES IN LEUKEMIA DIFFERENTIATION proliferation was assessed using a Coulter model ZBI particle counter urements, and the content of microtubules was measured by flow (Coulter Electronics, Hialeah, FL). HL-60 cell differentiation was cytometry as described above. quantified by the functional capacity to generate Superoxide, as meas ured by the reduction of NBT (Sigma). Cells in growth medium were RESULTS mixed with PBS containing 1% NBT and 1 ¿jg/mlof 12-0-tetradeca- noylphorbol 13-acetate (Sigma) in a 1:1 (v/v) ratio and incubated at HL-60 leukemia cells were induced to differentiate along the 37"C for 30 min. NBT positivity was measured by counting 100 to 200 granulocytic pathway by exposure to l /¿MRAfor 5 days. No cells and expressing the results as the percentage of NBT-positive cells. inhibition of growth was observed 3 days after the exposure to The expression of the mature myelocyte surface marker Mo 1 was RA compared to untreated control cells (Fig. la); significant also used as an additional measure of HL-60 cell differentiation. Cells (1 x 10*)were incubated with Mo 1-FITC monoclonal antibody (Coul inhibition of growth occurred, however, 4 and 5 days after ter Immunology) for 30 min on ice, and washed with PBS or fixed in PBS containing 3.7% formaldehyde for 30 min, and Mo 1 positivity 100 was measured using flow cytometry. The ability of cells to phagocytize particles was monitored as de «2.0 u »u scribed by Blair et al. (14) to serve as a third phenotypic marker of HL- | 15 160 60 cell differentiation. Briefly, 1x10* cells were incubated with 1-2 x ¡1.0 IO7 fluorescent microspheres of 1.75 ^m diameter (Polysciences Inc., Warrington, PA) for 24 h in a humidified 37°Catmosphere containing g 0.5 20 0.0 5% COj in air after exposure to RA for 4 days. Cells were then washed 234 4 5 twice with PBS and analyzed using a flow cytometer, and the percentage Time (Days) Time (Days) of cells containing FITC-microspheres was calculated. Fluorescence Microscopy of Cytoskeletal Components. The organi zation of MT and the IF, vimentin, was visualized by immunofluores- a so (j 60 cence microscopy. Control or RA-treated cells (2 x IO5cells/ml) were I 60 ÃŒ washed with PBS, resuspended in PM2G buffer, and 4 x IO4cells were _ 40 20 collected using a Cytospin II (Shandon Inc., Pittsburgh, PA) directly i 20 onto a 12- x 12-mm glass slide at 1000 rpm for 10 min. Prepared slides were fixed with 3.7% formaldehyde in PM2G buffer for 30 min. Fixed 4 5 Control Relinoic Acid cells were incubated with PBS and then with 0.1 M glycine in PBS for Time (Days) 5 min each. Cells were then extracted with 0.3% Nonidet P-40 (Sigma) Fig. 1. Effects of retinoic acid on the proliferation and differentiation of HL- 60 leukemia cells. Cell were treated with 1 (.M RA and, at the indicated times, in PBS for 10 min and washed with PBS twice. MT and IF were cell numbers (a). NBT positivity (/>). and Mo 1 positivity (c) were determined. visualized by staining cells with a mouse monoclonal anti-a-tubulin The percentage of phagocytic cells was measured 5 days after exposure to RA antibody and an anti-vimentin antibody, respectively, for 30 min fol (d). Points or bars, means of at least three separate experiments ±SE. lowed by rhodamine-conjugated goat anti-mouse immunoglobulin an tibody (Cappel, Malvern, PA) for 30 min. The stained slides were washed with PBS and then with distilled water before mounting using Q Control •¿1iiM Retinoic Acid Airvol 205 (Air Products and Chemical Inc., Allentown, PA) prepared as gelvatol as described by Jacobson et al. (15). All procedures were performed at room temperature. The slides were examined with a Nikon Optiphot microscope equipped with an epifluorescence attachment using the G-1A filter and Plan Apochromat xlOO oil objective. Pho tographs were taken using an UFX automatic exposure system (Nikon) with Kodak TMAX (ASA 400) film. Flow Cytometric Measurement of the Cytoskeleton Content. The content of MT; the MAPs, MAP2 and tau; and the IF, vimentin, was measured by flow cytometry after immunofluorescent staining. Cells (1-1.5 X 106/ml) were fixed in PM2G buffer containing 3.7% formal dehyde for 30 min. Fixed cells were incubated in PBS and then in 0.1 M glycine in PBS for 5 min each. Cells were then permeabilized by treatment with 10 Mg/ml of L-a-lysophosphatidylcholine (Sigma) in PBS for 30 min, washed with PBS twice, and resuspended in PBS containing 0.5% bovine serum albumin. Cells were stained with mouse monoclonal antibodies against a-tubulin (clone DM1 A), /3-tubulin (clone Tub 2.1), MAP2 (clone HM-2), tau (clone tau-2), and vimentin (clone Vim 13.2), obtained from Sigma, for 30 min, followed by FITC- conjugated goat anti-mouse immunoglobulin antibody (Cappel) for 30 min. Stained cells were analyzed using a Becton Dickinson flow cytom eter. The relative fluorescence intensity of cells stained with ami cytoskeleton antibodies was measured and compared to cells stained with secondary antibody alone. The relative fluorescence intensity of RA-treated cells was compared to that of untreated control cells and expressed as a percentage of the control fluorescence. Retinoic Acid ( Induction of Microtubule Depolymerization and Polymerization. In Fig. 2. Comparative effects of retinoic acid on the proliferation and differen experiments in which microtubules were disassembled, cells were in tiation of HL-60, HL-60/S4 supersensitive, and HL-60/R3 resistant clones. HL- cubated with 2 /IM (Sigma) for 1 h. To promote microtubule 60/S4 cells were treated with 0.01. 0.1, or 1 «\tRA, and HL-60/R3 cells were polymerization, cells were treated with 0.5 UMtaxol, obtained from the treated with l UMRA for 4 days; cell numbers (a). NBT positivity (¿>and

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. CYTOSKELETON CHANGES IN LEUKEMIA DIFFERENTIATION treatment with the retinoid. The ability to generate Superoxide treated with 1 ¿IMRAexhibited 24% more inhibition of growth as determined by the reduction of NBT, the expression of the than HL-60 parental cells exposed to the same concentration mature myelocyte surface marker Mo 1, and the capacity to of retinoid (Fig. 2a). In contrast, no inhibition of growth was phagocytize microspheres were used as measures of the state of observed in RA-resistant HL-60/R3 cells treated with the reti maturity of HL-60 cells. No NBT or Mo 1 positivity occurred noid. The effects of exposure to 0.01, 0.1, and 1 ^M RA for 4 on the initial 2 days after exposure to RA; however, NBT days on the NBT positivity (Fig. 2b) and the Mo 1 positivity positivity (Fig. 1¿>)andMo 1 positivity (Fig. le) were observed (Fig. 2c) of HL-60 parental and HL-60/S4 cells were measured. by 3 days and continued to increase through 5 days after NBT and Mo 1 positivities were concentration dependent in initiation of the maturation process. The kinetics of NBT and both parental and HL-60/S4 supersensitive cells. A higher Mo 1 positivity of the population of HL-60 cells undergoing percentage of NBT- and Mo 1-positive cells were produced in maturation were similar, indicating that the required for HL-60/S4 cells than in HL-60 parental cells at all of the these functional properties were activated in a complementary concentrations of RA tested. In contrast, RA-resistant HL-60/ fashion. Furthermore, the phagocytic capacity of HL-60 cells R3 cells treated with 1 MMRA for 4 days exhibited only 9% increased by 46% over that of untreated control cells following NBT positivity (Fig. 2d) and 15% Mo 1 positivity (Fig. 2e). No treatment with RA for 5 days (Fig. id). difference was observed between HL-60 parental cells and HL- HL-60/S4 supersensitive and HL-60/R3 resistant clones 60/S4 cells in their ability to phagocytize particles 5 days after were treated with up to 1 /¿MRAfor 4 days, and RA exposure, whereas the percentage of phagocytic HL-60/R3 and differentiation were determined (Fig. 2). HL-60/S4 cells cells was significantly less (23%) than that of wild-type HL-60

Fig. 3. Effects of retinoic acid on the cytoskeletal organization of HL-60 leukemia cells. Untreated cells (a and c) and cells treated with 1 ,/M RA (b and d) for 4 days were processed for microtubules (a and b) and vimentin intermediate filaments (c and d) using fluorescent staining as described in "Materials and Methods." Bar, lOnM. 951

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vimentin compared to those stained with FITC secondary an 750 - tibody alone was 106, 25.2, 7.2, 6.7, and 5.7, respectively. To

Ja 50C-§Z= ensure the effectiveness of the methodology used to measure vS\ the content of cytoskeletal components, HL-60 cells were iA l treated with 2 ^M colchicine (an MT-disrupting drug) or 0.5 250-/-..' '!/'-/ fiM taxol (an MT-aggregating agent) for l h and processed for V-'IO flow cytometric analysis. Disruption of MT after exposure to 10 10 10 10 10!FlTC/fl-Tubulind750IO1 |0! colchicine was accompanied by an 88% decrease in the content FlTC/a-Tubulin of MT, as compared to that of untreated cells. In contrast, treatment of HL-60 cells with taxol resulted in a 78% increase -JÕ in the content of MT measured by this methodology (data not shown). 500-EZ= \/*\ The content of MT; the MAPs, MAP2 and tau; and the IF, ''*I vimentin, of HL-60, HL-60/S4, and HL-60/R3 cells was quan 250-0J- \v\' i\/ tified after induction of differentiation by RA. Control HL-60 ^' V \. cells had relatively constant levels of MT (Fig. 5, a and b), 10° IO1 IO' 10' 10 IO' IO 10 MAP2 (Fig. 6a), tau (Fig. 6¿>),andvimentin (Fig. la) for 3-4

FITC/MAP2 FITC/tau days after growth in culture; a slight decline in the concentration of these cytoskeletal components then occurred in untreated cells. In contrast, cells exposed to 1 MMRA expressed more than a 100% increase in the content of MT 4 days after the retinoid, while the content of MT declined sharply at day 5 (Fig. 5, a and b). The increase in the content of a- and /3-tubulin g »H in HL-60 parental cells also occurred in the RA-supersensitive clone, HL-60/S4, after exposure to RA for 4 days and was concentration dependent, with HL-60/S4 cells having a greater 10 IO1 IO 10 increase in the concentration of MT than parental cells (Fig. 5, FITC/Vimentin c and d). In contrast, the RA-resistant clone, HL-60/R3, had a Fig. 4. Immunofluorescent distribution of HL-60 leukemia cells stained with anti-cytoskeleton antibodies. HL-60 cells were fixed with formaldehyde, pernio significantly lower increase in the content of MT than parental abilized with lysophosphatidylcholine, fluorescent stained using anti a-tubulin (a), 0-tubulin (b), MAP2 (c), tau (cytoplasm (Fig. 3a). In contrast, the MT of cells exposed to RA were stained with higher intensity, and the MT were organized into bundles (Fig. 3Ä). IF of vimentin were not readily visible in untreated HL-60 cells (Fig. 3c); however, the vimentin content of cells exposed to RA for 4 days was clearly observable as structures organized into a filamentous network (Fig. 3d). 0 1 The amount of cytoskeletal components was quantified using Retinoic Acid ,.M i flow cytometry after HL-60 cells were exposed to RA, fixed, Retinoic Acid ( .Al Fig. 5. Effects of retinoic acid on the content of microtubules during the permeabilized with lysophosphatidylcholine, and immunoflu- maturation of HL-60 leukemia cells. For the time-course study (a and b), HL-60 orescent stained with monoclonal antibodies specific for partic cells were treated with 1 J/M RA and, at the indicated times, the content of MT ular cytoskeletal components. The fluorescent intensity of cells was measured by fluorescent staining using anti-a-tubulin (a, c, and e) or ami ,f~ tubulin (b, d, and/) antibodies and analysis by flow cytometry. For concentration stained with anti-cytoskeleton antibodies, compared to those studies, the RA-supersensitive clone, HL-60/S4 (c and

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Control to quantify the changes in these proteins during cellular growth 200 Retinole Acid and differentiation.

i 150 An increase in the contents of MT and vimentin was observed in HL-60 cells following exposure to RA for 4 days. The increase in the content of MT was accompanied by a transient increase in the amounts of MAP2 and tau during the course of 50 the maturation process. That the changes in MT and vimentin 1234 234 Time (Days) Time (Days) are associated with the maturation process was supported by the measurement of these cytoskeletal components in RA- Fig. 6. Effects of retinoic acid on the content of MAPs during the maturation supersensitive and -resistant clones and by the finding that of HL-60 leukemia cells. Cells were treated with 1 >IMRA and, at the indicated times, MAP2 (a) and tau (¿)were measured by fluorescent staining using anti- induction of the differentiation of HL-60 cells by dimethyl MAP2 or anti-tau antibodies and analysis by flow cytometry. The contents of sulfoxide is accompanied by an increase in the content of MT MAP2 and tau were expressed as percentages of that of untreated control cells at day 4. Points, means of at least three separate experiments ±SE. (data not shown). It is not clear whether the changes in the cytoskeleton that were observed represent modifications in the metabolic scaffolding of the cell as a result of the attainment of HL-60 cells after exposure to 1 ¿¿MRAfor 4 days (Fig. 5, e and a mature phenotype or are involved in the initiation of the /). Similar changes in the contents of MAP2 (Fig. 6a), tau (Fig. induction process. The functions of mature neutrophils, such 6ft), and vimentin (Fig. 7) were also produced in HL-60, HL- as phagocytosis (16), release of proteinases after chemotactic 60/S4, and HL-60/R3 cells following exposure to RA. stimulation (17), and the oxidase involved in the respiratory burst (18), have all been shown to be associated with F-actin DISCUSSION and the actin-binding network. Whether MT and vi mentin are also involved in these functions is not known. We Induction of leukemia cell differentiation has been reported have shown that the kinetics of the expression of the content to be accompanied by changes in the cytoskeleton (9). Changes of MT during HL-60 leukemia cell maturation do not precisely in MT, determined by electron microscopy (3), and alterations correspond to the attainment of markers of the granulocyte in the IF, vimentin, determined by immunofluorescence mi mature phenotype, implying that these changes are not the croscopy (11, 12) of HL-60 cells induced to differentiate along result of the mature state. In this regard, MT have been shown the granulocytic or monocytic pathways, have been shown. to interact with components of the cellular Furthermore, alterations in the expression of several cytoskel- pathways (19, 20) and to be associated with a cellular trans eton-associated proteins, which were characterized by their forming protein and a tumor suppressor (21). resistance to detergent extraction, during granulocytic and monocytic maturation of HL-60 cells, determined by two-di Control mensional gel electrophoresis, has also been described (8), but 250 Retinoic the identity of these proteins, except for vimentin, is unknown, 200 and the kinetics of the changes in these cytoskeletal components 150 during the maturation process have not been characterized. To evaluate in depth the changes in cytoskeletal components that occur during the maturation of HL-60 cells, we used monoclo 50 nal antibodies against specific cytoskeletal components and O 1234 5 6 flow cytometry to measure by immunofluorescence the content Time (Days) of cytoskeletal proteins. This approach was designed to measure cellular structural proteins involved in the cytoskeleton network and proteins associated with this network. The effectiveness of this approach was demonstrated by the correspondence of the MT content of HL-60 cells, determined by flow cytometry, with the expected changes produced by exposure to colchicine and taxol. Thus, disruption of MT by colchicine resulted in a decrease in the content of MT, and polymerization of the MT by taxol was accompanied by an increase in the cellular content of these structures, relative to untreated control cells. These Findings indicate that cellular soluble proteins were effectively removed by the technology used and that the monoclonal anti- cytoskeleton antibodies exhibited specificity in their capacity to bind to the cytoskeletal network. The same procedure is cur rently being used to measure changes in the content of F-actin and actin-binding proteins during HL-60 leukemia cell matu 0 1 ration. It should be noted that we have been successful in Retinoic Acid (iiM) measuring the content of the nuclear-associated oncogene pro Fig. 7. Effects of retinoic acid on the content of vimentin during the maturation of HL-60 leukemia cells. For the time-course study (a), HL-60 cells were treated teins, Jos and jun. as well as -phosphorylated proteins with 1 «\iRA and, at the indicated times, the content of vimentin was measured with this procedure using their respective monoclonal antibod by fluorescent staining using anti-vimentin antibody and analysis by flow cytom ies.-1Thus, with the ever increasing availability of monoclonal etry. For concentration studies, the RA-supersensitive clone, HL-60/S4 (ft), was treated with 0.01, 0.1, and 1 «IMRAand the RA-resistant clone, HL-60/R3 (c), antibodies to other cellular proteins, this procedure can be used was treated with 1 JIMRA for 4 days and compared to parental HL-60 cells. The *M.-F. Leung, J. A. Sokoloski, and A. C. Sartorelli, unpublished observation. content of vimentin was expressed as a percentage of that of untreated control cells at day 4. Points or bars, means of at least three separate experiments 'SI. 953

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10. Hass, R., Giese, G., Meyer, G., Hartmann, A., Dork, T., Kohler, L., Resch, Thus, MT may be involved in the transduction of signals during K., Traub, P., and Goppelt-Strube, M. Differentiation and retrodifferentia- the initiation of HL-60 cell differentiation. Further study of the tion of U937 cells: reversible induction and suppression of intermediate changes in the cytoskeletal network during leukemia cell differ filament protein synthesis. Eur. J. Cell Biol., 51: 265-271, 1990. 11. Dellagi, K., and Brauet, J. Alteration of vimentin intermediate filaments entiation may well lead to further understanding of the role of expression during differentiation of HL-60 and U-937 human leukemic cell these structures in the maturation process. lines. Leuk. Res., «.-611-616, 1984. 12. Hass, R., Bartels, H., Topley, N., Iladani. M., Kohler, L., Goppelt-Strube, M., and Resch, K. TPA-induced differentiation and adhesion of U937 cells: ACKNOWLEDGMENTS changes in ultrastructure, cytoskeletal organization and expression of cell surface antigens. Eur. J. Cell Biol., 48: 282-293, 1989. We would like to thank Rocco Carbone for his assistance with flow 13. Catino, J. J., and Miceli, L. A. Microtiter assay useful for screening of cell- differentiation agents. J. Nati. Cancer lust.. 80: 962-966, 1988. cytometric analyses. 14. Blair, O. C., Carbone, R., and Sartorelli, A. C. Differentiation of HL-60 promyelocytic leukemia cells: simultaneous determination of phagocytic ac tivity and distribution by flow cytometry. Cytometry, 7: 171-177, REFERENCES 1986. 15. Jacobson, K., O'Dell, D., Holifield, B., Murphy, T. L., and August, J. T. 1. Albert, B., Bray, D., Lewis, J., Raff, M., Roberts, C, and Watson, J. D. The Redistribution of a major cell surface glycoprotein during cell movement. J. cytoskeleton. In: B. Albert, D. Bray, J. Lewis, M. Raff, C. Roberts, and J. Cell Biol., 99:1613-1623,1984. D. Watson (eds.), Molecular Biology of the Cell, pp. 549-610. New York: 16. Stessei, T. P. The mechanical responses of white blood cells. In: ¡.I. Gallin, Garland Publishing, Inc., 1989. I. M. Goldstein, and R. Snyderman (eds.). Inflammation: Basic Principles 2. Bershadsky, A. D., and Vasiliev, J. M. Cytoskeleton. New York: Plenum and Clinical Correlates, pp. 325-342. New York: Raven Press, Ltd., 1988. Press, 1989. 17. Schettler, A., Thorn. H., Jockusch B. M., and Tschesche, H. Release of 3. Brown, W. J., Norwood, C. F., Smith, R. G., and Snell, W. J. Development proteinases from stimulated polymorphonuclear leukocytes. Evidence for of capping ability during differentiation of HL-60 human promyelocytic subclasses of the main types and their association with cytoskeletal leukemia cells. J. Cell. Physiol., 706: 127-136, 1981. components. Eur. J. Biochem.. 197: 197-202, 1991. 4. Hoffman-Lieberman, B., and Sachs, L. Regulation of actin and other proteins 18. Woodman, R. C., Ruedi, J. M., Jesaitis, A. J., Okamura, N., Quinn, M. T., in the differentiation of myeloid leukemic cells. Cell, 14: 825-834, 1978. Smith, R. M., Curnutte, J. T., and Babior, B. M. Respiratory burst oxidase 5. Nagata, K., Sagara, J., and Ichikawa, Y. Changes in contractile proteins and three of four oxidase-related polypeptides are associated with the cyto during differentiation of myeloid leukemia cells. I. Polymerization of actin. skeleton of human neutrophils. J. Clin. Invest., X": 1345-1351, 1991. J. Cell Bio!., 85: 273-282, 1980. 19. Zor, I . Role of cytoskeletal organization in the regulation of adenylate 6. Meyer, W. II.. and Howard, T. H. Changes in actin content during induced cyclase-cyclic adenosine monophosphate by hormones. Endocr. Rev., 4: 1- myeloid maturation of human promyelocytes. Blood, 62: 308-314, 1983. 21, 1983. 7. Kwiatkowski, D. J. Predominant induction of and actin-binding 20. Rasenick, M. M., O'Callahan, C. M., Moore, C. A., and Kaplan, R. S. GTP- protein during myeloid differentiation. J. Biol. Chem., 263: 13857-13862, binding proteins which regulate neuronal adenylate cyclase interact with 1988. microtubule proteins. In: M. De Brabander and J. De Mey (eds.), Microtu- 8. Bernai, S. D., and Chen, L. B. Induction of cytoskeleton-associated proteins bules and Microtubule Inhibitors, pp. 313-323. Amsterdam: Elsevier Science during differentiation of human myeloid leukemic cell lines. Cancer Res., 42: Publishers B. V., 1985. 5106-5116,1982. 21. Maxell, S. A., Ames, S. K., Sawai, E. T., Decker, G. L., Cook, R. G., and 9. Bernai, S. D., and Stahel, R. A. Cytoskeleton-associated proteins: their role Bute!, J. S. Simian virus 40 large T antigen and p53 are microtubule- as cellular integrators in the neoplastic process. CRC Crit. Rev. Oncol. associated proteins in transformed cells. Cell Growth Diff., 2: 115-127, Hematol., J: 191-204, 1985. 1991.

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Mun-Fai Leung, John A. Sokoloski and Alan C. Sartorelli

Cancer Res 1992;52:949-954.

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