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HL60) Variants Insensitive to Phorbol Ester Tumor Promoters1

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HL60) Variants Insensitive to Phorbol Ester Tumor Promoters1 [CANCER RESEARCH 44, 3280-3285, August 1984] Analysis of Human Promyelocytic Leukemia Cell (HL60) Variants Insensitive to Phorbol Ester Tumor Promoters1 Deborah W. Mascioli and Richard D. Estensen2 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455 ABSTRACT promoter (11). It is thought that this involves "tumor progression." Thus, while kinase activation may explain short-term effects of The cells of the human promyelocytic leukemia cell line (HL60) promoters (examples of short-term effects would be enzyme stop growing and differentiate into macrophage-like cells when induction or differentiation or stimulation of cell division), long- exposed to nw concentrations of the phorbol ester tumor pro term effects which involve "tumor progression" may involve other moter 12-O-tetradecanoylphorbol-13-acetate (TPA). By exposing mechanisms such as changes in chromosomal composition. cells to the frameshift mutagen ICR-191 and subsequently se While such changes may well involve C kinase, proof may be lecting for resistance to the differentiating effects of nM amounts difficult. Isolation and analysis of cells resistant to TPA may be of TPA, we have isolated TPA-insensitive variants. These var useful in understanding the biochemistry of promotion. iants can grow in up to 320 nw TPA concentrations and do not For this study, we used the human tumor cell line HL60. HL60 differentiate into morphologically or functionally mature macro cells are a continuously proliferating human promyelocytic cell phages. The number of phorbol ester receptors, their affinity for line described by Gallagher et al. (15) derived from the peripheral phorbol dibutyrate, and the regulation of receptors are the same blood of a patient with acute promyelocytic leukemia. HL60 cells as for wild-type HL60 cells. As the resistance to TPA increases can move along 2 separate lines of hematopoietic differentiation. in the variants, so does the number of cells with increased ploidy. In the presence of such inducers as DMSO (8), retinoic acid (5), Wild-type HL60 cells are nearly 100% hypodiploid with a modal dimethyl formamide (8), and L-ethionine (22), HL60 cells differ chromosome number of 43, while a partially TPA-resistant variant entiate into neutrophils and develop complement receptors, be (DM30) has 30% hyperdiploid cells with a mean chromosomal come phagocytic as well as chemotactic, and show respiratory number of 70, and a completely resistant variant (DM90) is 93% burst activity (7). In response to 1.6 nM TPA, HL60 cells stop hyperdiploid averaging 74 chromosomes/cell. The variants dif growing and differentiate into macrophage-like cells (27). We ferentiate into neutrophils in response to dimethyl sulfoxide but report the isolation of variants which are insensitive to the are defective in respiratory burst activity as assayed by the growth-inhibitory and differentiation-inducing effects of 1.6 nM reduction of the dye nitroblue tetrazolium. These variants could TPA. There is no statistically significant change in phorbol dibu be useful in determining the mode of action of TPA in the tyrate receptor number, avidity, or regulation in these variants. promotion of tumors. The variants show an increase in ploidy as their resistance to TPA-induced effects increases. The variants continue to differ INTRODUCTION entiate in response to DMSO, but DMSO-treated cells do not demonstrate the usual response to inducers of respiratory burst. Tumor promoters produce many effects that both activate and inhibit biochemical processes at the level of the membrane, MATERIALS AND METHODS cytoplasm, and nucleus (11, 29). Thus, they have been likened to hormones (4). Most recently, the direct activation by TPA3 of Chemicals. TPA and PDB were obtained from CCR, Inc. (Eden Prairie, a platelet protein kinase, "C" kinase (6), has been observed, and MM). Stock solutions were made in ethyl acetate and stored frozen for a phorbol dibutyrate binding "receptor" copurifies with the C not longer than 2 months. At the time of use, the ethyl acetate was evaporated, and dilutions were made into DMSO and growth medium. kinase (24). The understanding of the kinase and its interactions ICR-191 (Polysciences, Inc., Warrington, PA) was stored at 4°in the may well clarify many of the actions of promoters; however, dark and dissolved in DMSO immediately before use. [20-3H]PDB (7.6 some other observations may not fit neatly with that finding. Ci/mmol) and [3H]TPA (8.0 Ci/nmol) were obtained from CCR Inc., in Several workers have observed chromosomal abberations with ethanol:toluene:ethyl acetate. Aliquots were evaporated under nitrogen TPA treatment. These have included aneuploidy, both hypo- and immediately before use and redissolved in DMSO and growth medium. hyperploidy, as well as chromosome breaks and rearrangements The final concentration of DMSO was always less than 0.1% in all (3, 12). The suggestion has been made that some of the pro experiments. moting effects of TPA may be mediated through oxidative mech Cell Culture. HL60 cells were obtained from Dr. Steven Collins and anisms that produce DMA damage through the production of Dr. Robert Gallo and were routinely cultured in 75-sq cm tissue culture free radicals (13). flasks (Falcon), RPMI 1640 plus 10% fetal bovine serum (Flow Labora The basic phenomenon of tumor promotion is one that takes tories, McLean, VA), and gentamicin (50 MQ/ml; Grand Island Biological a long period of time as well as repeated applications of the Co., Grand Island, NY). For monitoring growth, cells were plated at 50,000 cells/well in 24-well tissue culture dishes (Falcon) and counted in 1This work was supported by USPHS Grant CA22195 and the Elsa U. Pardee triplicate at varying intervals over 10 days in a Model ZBI Coulter Counter. Foundation. To obtain subclones, exponentially growing cells were plated at 1 cell/ 2 To whom requests for reprints should be addressed. well in 96-well tissue culture desks (Linbro, McLean, VA) in RPMI 1640 3 The abbreviations used are: TPA, 12-O-tetradecanoylphortooH 3-acetate; DMSO, dimethyl sulfoxide; PDB, phorbol-12,13-dibutyrate; NBT, nitroblue tetra plus 20% fetal calf serum. Treatment with Mutagens. Exponentially growing cells (10e cells) zolium; SOD, Superoxide dismutase. Received May 19, 1983; accepted May 3, 1984. were treated with ICR-191 (2.5 j<g/ml) for 24 hr in the dark. This dose 3280 CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. ,,. x ._....„..,, Analysis of TPA-resistant HL60 Variants has beendeterminedto yield approximately 10% survival.The cells were mutagenic to diploid human lymphoblasts (10). HL60 cells were then washed once with phosphate-buffered saline, resuspendedin fresh treated with ICR-191 (see "Materials and Methods") and then medium,and allowed to grow back over a period of 6 to 7 days to allow subcultured three times in 1.6 nw TPA. These subcultures con for phenotypic expression. sisted of treating HL60 cells for 24 to 48 hr with TPA, harvesting Morphologicaland HistochemicalStains. Morphologywas deter minedon a minimumof 200 cells stained with Wright-Giemsa;a-naphtyl the unattached cells, and repeating the cycle. Attempts to isolate TPA-resistant variants by a single cycle of growth in concentra acetate activity was determined using Sigma Kit 90. NBT reduction was measuredusing Sigma Kit 840 (Sigma Chemical Co., St. Louis, MO). tions of TPA 1.6 nw or greater proved unsuccessful. Of several [20-3H]PDBBinding Assay. The bindingsare carriedout in 1.5-ml hundred HL60 clones isolated in response to this repeated polypropylene microcentrifuge tubes (Fisher, Chicago, IL). Each tube cycling, a few were resistant to the growth-inhibitory and/or contains 106 cells (precooled to 4°for equilibrium binding) and [20- differentiation-inducing effects of TPA at greater than 1.6 nw 3H]PDBin a final volume of 0.25 ml in RPM11640 (serum-free)plus BSA concentrations. One variant (DM30) showed about a 10-fold (1 mg/ml). To measure nonspecific binding, 30 I¡MunlabeledPDB are difference in its growth suppression by TPA (Chart 15) as added. All points are done in triplicate. The tubes are allowed to equili compared to wild-type HL60 (Chart 1A). DM30 grows as well as brate overnight at 4°,andbinding is terminated by centrifugation (800 x g x 15 min). The supernatants are,sampled for unbound [20-3H]PDB the untreated controls up to 1.6 nw TPA. DM30 will still grow slowly in 4.8 to 16 nM TPA. The latter concentrations completely and are carefully aspirated, and the pellets washed once with cold inhibit growth of wild-type HL60s. A variant derived from DM30 phosphate-buffered saline. The tips of the tubes (containing the cell pellet)are cut off and dropped into 3-ml scintillationvials containing0.25 by continued growth in 8.0 nw TPA, DM90 was even more ml of 0.5% Triton X-100 and allowed to solubilize for 2 hr at room resistant to TPA (Chart 1C). temperature.Aquasol(New EnglandNuclear,Boston, MA)is then added, When TPA-treated DM30 cells are analyzed for morphological and the radioactivity is counted. Specific [20-3H]PDBbound is defined (Chart 2A) and functional maturity, the presence of the monocyte as the total amount of [20-3H]PDB bound minus the amount bound in enzyme a-naphthyl esterase (Chart 26), only 50 to 60% of the the presenceof 30 /<MunlabeledPDB. cells differentiated by these criteria. DM90 cells (data not shown) Cytogenetic Analysis. Midlogarithmicphasecells (16 x 106 total) did not mature functionally or morphologically in up to 320 nw were treated with Colcemid (60 ng/ml; demecolcine; Sigma) for 1 to 2 doses of TPA. There were no esterase-positive cells and only 3 hr, centrifuged at 200 x g for 5 min and resuspended in 5 ml of freshly made 0.075 M KCI for 20 min at 37°.Thecells were again centrifuged to 4% monocytoid cells.
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