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 4Â°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

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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 4Â°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 IÂ¡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 4Â°,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 /

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106 100 Control

100r

0.51

Chart 2. Morphological and functional differentiation of HL60 (â€¢)andDM30 (O) cells treated with varying concentrations of TPA for 5 days. For morphology (A), cells were stained with Wright-Giemsa and scored for the presence of monocytoid cells. Functional differentiation was measured as the percentage of cells staining positively for the monocylic enzyme a-naphthyl esterase.

functional and morphological differentiation. HL60, DM30, and DM90 consisted of almost 100% mature cells (metamyelocytes CD and neutrophils) after 5 days in DMSO. HL60 and DM30 cells U were also positive for cell surface complement receptors, char acteristic of phagocytic cells (Table 1) (21). DM30 cells, however, were deficient in respiratory burst activity, another characteristic of phagocytic cells. As shown in Table 1 and Chart 5, DM30 had one-half to one-third as many cells capable of reducing NBT as HL60. DM90 (Table 2) shows about one-tenth as many NBT- positive cells as HL60. This defect was not specific to the TPA- sensitive receptor for respiratory burst and extended to other activators of respiratory burst as well. The proportion of DM30 cells reducing NBT in response to both zymosan-activated serum 160nM and concanavalin A was about 3-fold less than HL60 (Table 1). \ 80nM. 320nM â€¢Control,32nM Chromosomal Studies. HL60 cells have been reported to be 16nM near diploid with 45 or 46 chromosomes (15). The chromosome number of our wild-type HL60 cells is 43 or 44. Chromosomal spreads of DM30 revealed that 30% of the cells were polyploid with an average number of 70 chromosomes. The remaining 70% of the cells had an average number of 43 chromosomes. Results from another laboratory (20) indicated that a TPA-insensitive HL60 variant (HL60TR) was tetraploid. This variant was 1.5% DMSO obtained by culturing in low TPA concentrations (20). This sug gested to us that polyploidization might be induced by repeated culturing in TPA and might be a general mechanism for gener ating TPA-resistant HL60 variants. To test this hypothesis, DM30 was cultured in 8 nMTPA for 4 months. At the end of this period, these DM30 cells, now referred to as DM90, were examined for their chromosomal and DNA content. 10 DM90 cells were 92% hyperdiploid with an average number 8 of 74 chromosomes. Analysis of the DNA content of DM30 and Time (days) DM90 relative to HL60 by flow cytofluorometry revealed a sig Chart 1. Growth of HL60 (A) and variants DM30 (B) and DM90 (C) in varying nificant shift of the DM90 cells to a near tetraploid (4N) DNA concentrations of TPA (â€¢)and1.5% DMSO (O). content (results not shown). This shift in DNA content of DM90

3282 CANCER RESEARCH VOL. 44

12Â°Â°rA 1000p

1000

800 ? 500-

600

400

5 10 30 45 60 200 05 10 Bound (pmoO Time (minutes) 0 Chart 4. Down regulation of [20-'H]PDB binding by HL60 (â€¢)and DM30 (O) cells at 37Â°.The bindings were performed with 17.1 nM 120-'H |PDB in the presence or absence of 30 MMunlabeled PDB. Specific binding is per 10* cells. 1400 mO g 1200 Table 1 Ã–L Functional differentiation of HL60 and DM30 in response to DMSO <Â¿, 1000 Cells were treated with 1.25% DMSO for 6 days. HL60 DM30 g 800 % of cells reducing NBT In response to various inducers of respiratory burst o. O) Inducer TPA (50 ng/ml) 39.5 Â±2.1 " 18.5 Â±0.7 I 600 o Zymosan (1 mg/nH) 24.0 Â±1.4 9.0 Â±1.4 ConcanavaHn A (50 ng/ml) 40.0 Â±1.4 9.5 Â±0.7 400 None O O % of complement receptor-positive cells 200 Treatment None 0 0 DMSO 31.0 Â±4.2 32.5 Â±0.7 O * Mean Â±S.D.

600

006 400 Â¿i 004 li Â°Â°2:\0 200 01 05 .10 '88(pmoO11Bound 1 0^C7rii0 10 27- 122 183 fyjPDB (nM) Charts. Equilibrium binding of [20-'H]PDB to HL60 (A), DM30 (8), and DM90 (C). Cells were incubated for 24 hr at 4Â°with varying concentrations of |20-3H |PDB in the presence or absence of 30 MM unlabeled PDB. Specific binding is per 10* cells, inset, Scatchard analysis of binding data. A, k, = 20.2 Â±17.3 nM (S.E.); R, = 94,561 Â±17,209 binding sites/cell; r = 0.86; p < 0.02. B, kâ€ž=18.1 Â±10.93 nM; R, = 78,901 Â±25,865 binding sites/cell; r = 0.86; p < 0.01. C, K,, = 16.3 Â±5.3 HM; a = 49,000 Â±24,807 binding sites/cell; r = 0.93; p < 0.01 (S.E. based on data from 3 separate experiments). Â®.best straight line as determined by linear regression analysis. was accompanied by complete resistance to the growth-inhibi tory effects up to 320 OM(200 ng/ml) (Chart 1C). SOD. SOD is a scavenger of Superoxide aniÃ³n radicals (O2~) and has been previously shown to completely inhibit TPA-stim- ulated 02~ production in HL60 cells (21). SOD levels in both 10 25

untreated and DMSO-treated HL60, DM30, and DM90 cells were TPA (ng/ml) measured. The results are summarized in Table 2. While unin- Chart 5. TPA-induced respiratory burst activity in HL60 (â€¢)andDM30 (O) cells caused to differentiate by treatment with 1.5% DM30 for 7 (A) and 5 (S) days. Cells duced HL60, DM30, and DM90 cells had essentially the same responding to TPA with respiratory burst reduced the dye NBT. A minimum of 500 amount of activity, DMSO-treated DM30 cells had approximately cells were scored for each determination. 8 times and DM90 about 14 times more SOD activity than did HL60 cells. while DM90 had 10%, DM30 20%, and HL60TR 7%. HL60TR, the tetraploid variant of Acs and Christman,4 also Metabolism of [3H]TPA by HL60 or DM30. While O'Brien ef did not show a decrease in SOD activity in response to DMSO al. (25) have described metabolism of (3H]TPA in cultures of and had enzyme levels similar to that for DM90 (data not shown). mouse cells, examination of extracts of HL60, DM30, plus DM90 All the aforementioned variants are defective in the NBT re variant cultures exposed to [3H]TPA for up to 48 hr failed to sponse to TPA. DMSO-treated HL60 contained 78% NBT+ cells, reveal any significant metabolism of [3H]TPA. The extracts of

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Taue 2 (17,18), in a study of nonmetastatic mouse tumor cell variants, Respiratory burst and SOD activity in uninduced and DMSO-treated cells found that as these cells became highly metastatic, they also of NBT*cells0 CelltypeHL60 SOD/mgprotein11 became polyploid. There is a parallel between the time required 8.0 Â±21.9" HL60-DMSO" for production of polyploid cells by TPA in our hands and that 4.4 Â± 2.2 71.7 Â±5.7 DM30 165.0 Â±29.7 0 for tumor promotion. It is possible that the process of promotion DM30-DMSO" 34.4 Â± 8.6 20.7 Â±2.1 induces and/or selects polyploid tumor cells that are resistant to DM90 109.0 Â±15.6 0 the differentiating effects of TPA and that at least a portion of DM90-DMSCTUnits 56.7 Â± 6.6% 9.0 Â±1.0 " Mean Â±S.D. promotion is due to an event associated with increases in ploidy. 6 The cells were treated with 1.25% DMSO for 5 days. A testable prediction of this hypothesis is that TPA-promoted tumors would be polyploid. media of both HL60 DM30 and DM90 cultures, analyzed as How TPA might act to produce polyploidization is unknown. It indicated in "Materials and Methods," revealed that 95% of the could be a "fusogen" causing membrane alterations such that [3H]TPA comigrated with cold authentic TPA. Known metabo cells would more readily fuse. TPA is known to have a variety of lites, i.e., 12-myristate and 13-acetate or phorbol alcohol, were effects on cell membranes (29). Alternatively, TPA could induce not detected (results not shown). polyploidization by disruption of mitosis. This might lead to nondisjunction or endoreduplication where the chromosomes DISCUSSION replicate without cell division. Endoreduplication in response to TPA has not been observed to date. We have isolated variants (DM30, DM90) of the HL60 human An unexpected phenotype of these variants was the apparent promyelocytic leukemia cell line which are resistant to growth- defect in functional maturation after DMSO-induced differentia inhibitory effects of the phorbol ester tumor promoter TPA. These tion. The variants have one-half to one-tenth of the cells respond variants continue to grow in the presence of TPA and do not ing with NBT reduction activity after exposure to TPA, zymosan, completely differentiate into macrophage-like cells. Their defect or concanavalin A. This phenotype is not due to a general defect in TPA responsiveness lies subsequent to the initial receptor of neutrophilic differentiation in response to DMSO, since all the binding event, since the variants contain normal numbers and variants show a normal increase in complement receptors, an responses of high-affinity phorbol ester receptors. Resistance is other marker of phagocytic capacity and differentiation, as well not due to metabolism since variants do not metabolize TPA. a smaller size and the morphological appearance of metamye- Perhaps the most interesting aspect of these variants is that locytes or neutrophils. One explanation may be a continued they become increasingly polyploid as their resistance to the production of the enzyme SOD which scavenges Superoxide growth-inhibitory and differentiation-inducing effects of TPA in aniÃ³nradicals and has been shown to suppress respiratory burst creases. The differences between wild type and variants in in HL60 cells (21). The variants have 8- to 14-fold more SOD expression of differentiated phenotype, both morphological and activity after differentiation with DMSO than normal HL60 cells. histochemical, are almost an exact parallel of percentage of This excess SOD could scavenge the O2~-radicals produced in polyploid DM30 cells. The number of cells that do not reduce response to TPA and thereby give rise to a lower number NBT parallels the number of cells that are polyploid in DM90. reducing the NBT dye. Why these cells continue to produce SOD Our variants were isolated by continuous subculturing in 1.6 and what relationship, if any, this has to the phenomenon of TPA nw and 8 nw doses of TPA. Efforts to isolate TPA-insensitive resistance are unknown. variants by a 3-day exposure to TPA has, to date, proved The experiments measuring NBT reduction in response to unsuccessful. Others have described TPA-insensitive HL60 var TPA on DMSO-differentiated HL60 cells and variants were orig iants isolated by longer-term exposure in TPA (16, 22). All of inally designed to test the question of whether or not the TPA these variants may constitute a common pathway to TPA resist signal was modified in the variants at a step beyond TPA binding. ance. The chromosomal composition of the variants of Huberman However, our NBT reduction endpoint was obscured by SOD. et al. (16) or Solanki ef al. (22) are not known, but the variant of Recent information4 indicates that while HL60TR does not re Christman and Acs4 is polyploid. spond to DMSO, it does respond to retinoic acid. Both DM30 Is polyploidy alone sufficient for these HL60 cells to become and DM90 also respond to retinoic acid and show NBT reduction resistant to TPA? Preliminary experiments using cytochalasin B like wild-type HL60 in response to TPA. This indicates that one to induce up to 60 to 70% binucleate HL60 cells indicate that process that is dependent on TPA binding and subsequent these type of polyploid cell is as sensitive to the growth-inhibitory transmission of the signal still intact in variant cells. Thus, not all effects of TPA as wild-type HL60s. Perhaps other chromosomal responses to TPA are inhibited in variants. alterations must occur as well for resistance to TPA to result, We hope that these variants will lead to a better understanding since doubling or tripling of gene dosage might not be expected of both short-term TPA action and its long-term effects. An to produce the several hundred-fold increase in the resistance alteration in some biochemical responses of these variants may to the growth-inhibitory effects of TPA that are seen in our most indicate which TPA effects are critical for short-term action. In resistant variant, DM90. addition, a common chromosomal abberation or rearrangement TPA is known to induce polyploidy in other eukaryotic cells. may be found in these polyploid-resistant cells indicating a pos Human lymphocytes, newborn mouse epidermal cells, and the sible long-term effect of TPA. yeast Saccharomyces cerevisiae all become polyploid in re sponse to TPA (12,13,26). Polyploidy, while infrequent in normal ACKNOWLEDGMENTS cells, is often found in tumors (30). An increase in chromosome number (from triploid to hypotetraploid) in tumors can be asso Our thanks to Janet V. Ditmanson for technical assistance and Donna Jacobson ciated with poor prognosis and tumor progression. Kerbel ef al. for preparation of this manuscript.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. Analysis of Human Promyelocytic Leukemia Cell (HL60) Variants Insensitive to Phorbol Ester Tumor Promoters

Deborah W. Mascioli and Richard D. Estensen

Cancer Res 1984;44:3280-3285.

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