Transcriptional regulation of the c-myc protooncogene by 1, 25 ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1987 by The American Society of Biologicel Chemists, Inc.

Vol. 262, No.9,Issue of March 25, pp 4104-4108 1987 Printed in ~ . S . A .

Transcriptional Regulation of thec-myc Protooncogene by1,25Dihydroxyvitamin D3 in HL-60 Promyelocytic Leukemia Cells* (Received for publication, September 15,1986)

Robert U. Simpson, Todd HSU, Dale A. Begley, BeverlyS . Mitchell$, and Babak N. Alizadeh From the Departments of Phurmacology, Molecular Genetics, and InternalMedicine; The University of Michigan, Ann Arbor, Michigan 48109

Exposure of HL-60 promyelocytic leukemiacells to calcitriol results ina decrease insteady-state levels of c-myc mRNA and induces cellular differentiation.We have asked whether calcitriol a has direct effect on the transcription of the c-myc gene.1,25-Dihydroxyvitamin D3(1,25-(OH)2Ds)decreased RNA elongation ina nuclear run-off transcription assay by 4 h after treatment. In the continuous presence of 1,25-(OH)&, HL60 cell transcription of c-myc was decreased by 38% at 4 h and was abolished by 48 h.In contrast, the transcriptionof&actin was not affectedby 1,25(OHhD3 treatment. The rate of transcription of c-myc and &actin was proportional to the number of nuclei and to time. Furthermore, specific hybridization of cmyc and B-actin RNA was a linear function of RNA input. After a 48-h treatment, the c-myc/&actin ratio was decreased by 80-100% at [s2P]RNA inputs ranging from2 to 20 X lo6 cpm/ml. These data temporally correlate inhibition of c-myc transcription with decreases in the steady-state levels of c-myc mRNA as assessed by Northern blot analysis. We conclude that the effect of 1,25-(OH)2D3 on c-myc expressionoccurs at the transcriptionallevel.

Calcitriol or 1,25-(OH)2D3has been shown to induce the differentiation of HL-60 human promyelocytic leukemic cells into monocyte-like cells (10-15). Recently, Reitzma et al. (11) observed that this differentiation was preceded by a specific 3- to 4-fold decrease in the steady-state mRNA levels of the protooncogene c-myc which was evident within 4 h of exposure to calcitriol. We and others have since confirmed these data (14-15). Since expression of the c-myc gene has been closely linked to cellular proliferation (16-18), there hasbeen intense interest in the study of its regulation. Other nonsteroidal inducers of HL-60 cell differentiation have been associated with both transcriptional and post-transcriptional ef(19,20). We have analyzed fects on the levels of c-myc mRNA the effect of 1,25-(0H),D3 on the transcription of the c-myc gene in HL-60 cells to determine whether this steroid acts at the transcriptional or post-transcriptional level. MATERIALS AND METHODS

Chernicals-1,25-(OH)2D3was the gift of Dr. M. Uskokovic of Hoffman-La Roche and was >95% pure by high performance liquid chromatography. Purity was confirmed by ultraviolet spectroscopy. All other compounds were reagent grade or better. Cell Culture Conditions-HL-60 cells were propagated continuously in RPMI1640 media (Irvine Scientific, Irvine, CA), 10% horse serum (Sigma), 100 units of penicillin, and 0.1 mg/ml of streptomycin The steroid hormone 1,%-(OH),D3 is believed to act at (Sigma). Time course studies were initiated a t cell concentrations of the cellular levelby mechanisms similar to those of more 2-3 X 10’ cells/ml. Calcitriol in ethanol was added to cell cultures to a final concentration of 50 nM. Cells to which the same volume of extensively characterized steroid hormones which, when ethanol was added were used as control. Cells were harvested a t bound to specific intracellular receptors, have direct effects indicated time pointsand washed once with ice-cold 20 mM Tris-HC1 on geneexpression (1-3). Intracellular 1,25-(OH),D3receptors (pH 7.6), 2 mM M&L, 3 mM CaC12,3 mM dithiothreitol, and 300 mM and physiologic functions have been demonstrated in numer- sucrose (buffer A). ous tissues including intestine, bone, malignant tumors, heart, Nuclear Transcription Assays-Nuclei were prepared by a modifiskeletal muscle, and brain andthe 1,25-(OH)2D3receptor has cation of the method of Stallcup and Washington (21). Washed cells been purified and characterized from both avian and mam- were resuspended in buffer A a t 1-2 X l@cells/ml and homogenized with 15 strokes in a type B Dounce homogenizer to which an equal malian tissues (3-7). In addition, 1,25-(OH)zD3has been volume of buffer B (buffer A 0.2% Nonidet P-40, v/v) had been shown to have anti-proliferative effects on both neoplastic added. Nuclei were centrifuged down at 1000 X g and washed once cells and stimulated human lymphocytes (8-14). The poten- with buffer C (25% glycerol, 36 mM HEPES, pH 7.6, 3 mM MgCl,, 7 tial for widespread and importantphysiological effects of 1,25- mM dithiothreitol, 170 mM KCl). The nuclei pellet was resuspended (OH)zD3other than those on calcium metabolism has led to in buffer C at 1 pg DNA/ml. Nuclei isolated as described were reproducibly intact andfree of visible cellular debris. a major interest in itsmolecular mechanism(s) of action. Nuclear transcription activity was determined by measurement of [32P]UTPincorporation into RNA transcripts elongated in vitro as *This research was supported by National Institutes of Health Grant CA36507, American Cancer Society Grant CH-325, and grants described by McKnight and Palmiter (22). Nuclear transcription assays were carried out in a transcription buffer composed of 20% from the Children’s Leukemia Foundation of Michigan andthe Cancer Research Institute of The University of Michigan. Part of glycerol, 25mM HEPES (pH7.6), 2 mM MgCl,, 2 mM MnC12,5.5 mM this work was presented at theApril 1986 meeting of the Federation dithiothreitol, 134 mM KCl, 1mM each of ATP, GTP, and CTP, and of American Societies for Experimental Biology in St. Louis, MO. 50 pCi of [3ZP]UTP(New England Nuclear, 3000 Ci/mmol) at 26 ‘C The costs of publication of this article were defrayed in part by the for 20 min. 10 pl of DNase (4 mg/ml) was added to the reaction payment ofpage charges. This article must therefore be hereby mixture and incubated at 26 ‘C for 15 min to terminate the reaction. marked “advertisement” in accordance with 18 U.S.C. Section 1734 40 pl of 10% sodium dodecyl sulfate, 20 pl of 0.1 M NazEDTA, 0.1% diethyl pyrocarbonate, and 30 pl of proteinase K (20 mg/ml) were solely to indicate this fact. added and incubated at 37°C for 1h. The reaction mixture was diluted $ Scholar of the Leukemia Society of America. The abbreviations used are: 1,25-(OH)~D3,calcitriol, 1,25-dihy- by adding 200 p1 of TNE buffer (0.1 M NaCl, 10 mM Tris-HC1 (pH droxyvitamin D3; HEPES, 4-(2-hydroxyethyl)-l-piperazineethane- 8.0),0.1 M Na2EDTA) and was extracted with phenol/chloroform. Nuclear RNA was collected by two successiveethanol precipitations. sulfonic acid; kb, kilobases.

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Calcitriol Regulation of c-myc Immobilization of cDNA and Hybridization-pBR322 control plasmid and plasmid containing the inserted humanc-myc (exons 2 and 3, 1.5 kb) (23) andchicken @-actincDNA (1.8-kb PstI fragment) (24) were linearized by digesting with PstI. DNA was quantitated by the fluorescence method described by Labarca et al. (25). Linearized DNA was treated with 1/10 volume of 3 M NaOH and boiled for 5 min, and the reactionwas neutralized with 1/10volume of 10.5 M NH,Ac. DNA was blotted onto nitrocellulose (7 pg/dot) by using a Bio-dot apparatus (Bio-Rad). Nitrocellulose filters were dried under vacuum a t 80 "C for 2 h. '?P-Labeled RNA from nuclear transcription experiments was suspended in hybridization buffer (50% dionized formamide, 4 X SSC, 5 X Denhardt's solution, 200 pg of yeast RNA/ml). Hybridizations were carried out a t a concentration of 8 X lo6 cpm/ ml for 2-3 days a t 37 "C. Filters were prehybridized in the same buffer for 16-24 h at 37 "Cbefore hybridization. After hybridization, filters were washed in 2 X SSC, 0.1% sodium dodecyl sulfate a t 55 "C for four 1-h periods. Filters were then mounted on paper and exposed to Kodak XAR-2 film a t -20 "C. Quantitative results were obtained by scintillation spectrophotometry. Northern Blot Analysis-Total cellular RNA wasisolated from HL-60 cells by guanidine hydrochloride extraction(26)and was separated on 1% agarose-6.7% formaldehyde denaturing gels (27). Samples were transferred to nitrocellulose paper and hybridized to human c-myc (23) or chicken @-actin (24) inserts which had been nick-translated with [a-"PIdCTP (3000 Ci/mmol, New England Nuclear) to a specific activity of l@ cpm/pg. Blots were washed and autoradiographed with intensifying screens. NET Reduction-NBT reduction assays, used to monitor monocytic differentiation, were performed as described by Matsui et al. (28). Briefly, 2 X lo6 cells/ml were incubated with an equal volume of 0.2% nitroblue tetrazolium and dissolved in phosphate-buffered saline with 200 ng/ml tetradecanoylphorbol acetate a t 37 "C in 40 min. Cells containing bluish-black formazan deposits were scored as nitroblue tetrazolium positive, and each point represents a count of 200 cells. Miscellaneous Assays-Protein was measured by the methods of Bradford (29).Radioactivity onfilters was quantitated by liquid scintillation spectroscopy in a Beckman LS8100 counter. RESULTS

Characterization of the HL-60 Nuclear Transcription System-An in uitro transcription assay was optimized as previously described (30). Briefly, we found that M$+ (2 mM) and Mn2+ (2mM) were required for RNApolymerase activity, and RNA biosynthesis was linearly dependent on concentration of nuclei. a-Amantin, a selective inhibitor of RNA polymerase

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FIG. 2. Northern RNA blot analysis of c-myc and &actin RNA levels in 1,25-(OH)2D3(50 nM)-treatedHL-60 cells. HL60 cells were seeded a t 3 X lo5cells/ml and treated with 50 nM 1,25(OH),D, or vehicle. At time pointsnoted, RNA was extracted from 5 X 10' cells by the guanidium isothiocyanate method as described. RNA was electrophoresed on formaldehyde-agarose gels and transferred to nitrocellulose membranes. Filters were successively probed with nick-translated c-myc (A) and @-actin( B )cDNAs.

11, was found to inhibit totalnuclear RNA polymerase activity by 60%. [32P]UTP-labeled,elongated transcripts from nuclei transcription reactionswere examined for specific hybridization to DNA-blotted dots. Initially, the dependence of dot intensity and cpm of ["PIRNA hybridization was character0 myc ized. At 0.5, 1, 3, 5 , 7, and 10 pg of c-myc DNA/spot the I actin hybridization signal was 33, 109, 119, 201, 169, and 173 cpm of [32P]RNA, respectively. The[32P]RNAinput for these mYc hybridization reactions was IO7cpm of RNA/ml of transcripactin tion buffer. RNA (cpm)hybridized to pBR322 control plasmid was subtracted from c-myc (cpm) containing plasmid-RNA mYc hybrids. The data reveal that DNA excess is reached at 5-7 actin pg of DNA/dot for the c-myc probe. We routinely employed 7 pg of DNA/dot. Analysis of &actin DNA dot saturation my? yielded similar results (data not shown). actin Experiments were performed to explore the relationship FIG. 1. Hybridization signal as a function of RNA input in between [a-"PIRNA inputand hybridization signal. We the nuclear transcription assay of c-myc and &actin genes in HL-60 cells. Nuclei from 5 X lo' cells were incubated for 20 min found that [Q-~'P]RNA input from 1-10 x lo6 cpm/0.5 ml with ["'PIUTP using the conditionsdescribed under "Materials and yielded a linear hybrid signal as determinedby autoradiograMethods." c-myc and @-actin (7pg of DNA/dot) f i l t e r s were hybrid- phy and scintillation counting of dot radioactivity (Fig. 1). ized with 1-10 X lo6 cpm/0.5 ml of [a-"PIRNA for 3 days. HybridiThese conditions permitted quantitativeappraisal of total czation and washing conditions were as described. c-myc and @-actin [CI-~P]RNAtranscripts areshown to yield increased autoradiographic myc and @-actinRNA transcripts. Northern Blot Analysis of RNA from HL-60 Cells-HL-60 intensity with increased ["PIRNA input over the range of RNA (210 x 10' cpm/ml) used. cells were exposed to 50 nM 1,25-(OH)2D3or ethanol, and a t

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Time (hrs) FIG.3. Time course of alterations in rate of transcription of c-myc and @-actingenes in response to 1,25-(OH)*D3(50 nM) treatment. A. autoradiograph of dotblot analysis. Nuclear transcription assays were carried out using 5 X 10' nuclei per time point and measured in control or 1,25-(OH)'D3-treated cells after 1, 4, 12, 15, 24, and 48 h. A filter containing c-myc, 6-actin, or control pBR322 plasmids were incubated with 5 X lo6cpm/0.5 ml ["PIRNA hybridization buffer for 2.5 days as described under "Materials and Methods." Filters were autoradiographed with enhancing screensfor 16 h a t -70 "C. B, quantitative analysis of dot blot filters by scintillogaphy. Data represent the percent change in cpm of [a-"PIRNA bound to p-actin or c-mycDNA (minus pBR332cprn)from 1,25(OH)'D3-treated as compared to control cells.

2.5 5.0 7.5 12.5 10.0 RNA i n p u t (cpm / 0.5 m l x

FIG. 4. A, relationship of [cY-~'P]RNA input to dot blot intensity of RNA from control and 1,25-(OH),D3 (50 nM)-treated (72 h) cells. Nuclei from 5 X 10' cells were assayed for nuclear transcription as described under "Materials and Methods," and [o-~'P]RNA (1-10 X lo6cpm/0.5 ml) from control and 1,25-(OH)'D3-treated cultures were hybridized with c-myc, &actin, and controlpBR322 DNA (7 qg/dot). Hybridization, washing, and autoradiographywere as described under "Materials and Methods." Data presented here are representative of hybridifour separate experiments. B, quantitation of [c~-~'P]RNA zation to filters containingc-myc and 6-actin immobilized cDNA by liquid scintillation spectroscopy. A75-90% decrease is notedfor myc/ actin ratio for 1,25-(OH)zD3 (50 nM, 72 h)-treated cultures relative to control cultures.

tical to those previously reported by Reitzma et al. (11)and others (14, 15) and representative of three experiments in our various times, total RNA were isolated and c-myc and @-actin laboratory. Nuclear Transcription in Control and 1,25-(0H)2D3-treated steady-state RNA levels were quantitated by Northern blot analysis (Fig. 2, A and B). The ethanol controlswere identical Cells-The optimized nuclear run-off assay was used to meato the zero time point of each experiment (data not shown). sure nuclear transcriptionactivity in nuclei isolated from HLDifferentiation of cells was confirmed using nitroblue tetra- 60 cells treated with 1,25-(OH)2D~ or vehicle (ethanol) for 1, zolium dye reduction assay. The amount of c-myc RNA is 4, 12, 15, 24, and 48 h. Cultures were maintained simultaneunchanged from control values a t 1.0 h after the addition of ously with those used for the analysis of c-myc and @-actin 50 nM 1,25-(OH)*D3, butdropstoapproximately 50% of RNA levels. Fig. 3A shows the results of a nuclear run-off control after4 h and decreases to 10-30% of control after 12- assay after hybridization with filters, containing c-myc and 48 h. Fig. 2B shows that 1,25-(OH)2D3 induction of HL-60 @-actinplasmids, and autoradiography. At 1-h post-treatment, detectable cell differentiation is not associated with changes in @-actin no differences in c-myc transcriptionratesare (50nM)-treated cells, RNA levels. Furthermore, Fig. 2, A and B, show that 1,25- between controland1,25-(0H)& (OH)PDRregulation of c-myc RNA levels is not a function of whereas after 4 h, there is a decrease of approximately 38% a general decrease in total RNA levels. These data are iden- (cpm/dot) in the transcription of the c-myc gene. Treatment

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(16). On the other hand, Grosso and Pitot showed that treatment of HL-60 cells with dimethyl sulfoxide and 12-0-tetradecanoylphorbol-13-acetatedecreased transcription of the cmyc gene after 5 days in culture (19). Steroid hormones have been shown to regulate gene expression by regulating both mRNA synthesis and degradation (34-39). The most studied example of steroid hormone-mediated alterations in gene transcription is the regulation of mouse mammary tumor virus by glucocorticoids (34). Yamamot0 and co-workers (34) have shown that glucocorticoids first interact with a receptor protein that possesses DNAspecific binding capacity; the steroid-receptor complex then induces transcription of the mouse mammary tumor virus gene by interaction with specific transcriptional regulatory elements proximal to thepromoter and gene. Glucocorticoids have also been shown to regulate a acid glycoprotein expression by altering post-transcriptional RNA processing (36). Vitamin D, is metabolized in the liver to 25-hydroxyvitamin D3 and then in the kidney to 1,25-(OH)&. The latter form is a steroid hormone that has recently been found to regulate cellular processes not previously recognized as dependent on vitamin D intake ormetabolism (1-4). Such processes include insulin release from pancreas, heart and skeletal muscle function, and regulation of malignant cell growth and differentiation. Tanaka et al. (12) characterized a receptor specific for 1,25-(OH)2D3in the human promyelocytic leukemia HL-60 cell line which had properties similar tothe mammalian intestinal receptor. It has long been assumed that 1,25(OH),& acts by activation of hormone receptors which then DISCUSSION alter the transcription of specific genes. Specific stimulus of It has been shown that 1,25-(OH)2D3reduces c-myc mRNA total mRNA synthesis by 1,25-(OH)zD3has been shown to levels in HL-60 cells within 4 h of exposure (11, 14, 15). The occur in intestine (1-4). Recently, 1,25-(OH)2D3has been effect of 1,25-(OH)2D3on c-myc mRNA levels was reported to precede the induction of differentiation markers such as shown to regulate prolactin and parathyroidhormone steadystate mRNA levels (38, 39). However, studies of the mechalysozyme secretion, nonspecific esterase activity, and the apnism of action of 1,25-(OH)2D3have been hampered by the pearance of specific surface antigens (11,14,15). The decrease paucity of gene products that are characterized for eliciting in levels of specific mRNA species in a cell could result from biological response. In the present study we show that the cdecreased transcription of the message, increased degradation myc protooncogene is a gene whose expression is transcripof synthesized mRNA, or decreased protection of mRNA tionally regulated. We previously reported that 1,25-(OH)2D3 species (31). We have shown here that the transcription rate (50 nM) inhibition of DNA synthesis in HL-60 cells required of the c-myc gene, as measured by in vitro nuclear transcripa period of time greater than 18 h (30).However, within 48 h tion assays, is decreased by 1,25-(OH)zD3inHL-60 cells. of treatment, significant inhibition of DNA synthesis was Furthermore, we have shown that 1,25-(OH)2D3 treatment achieved (30). Data here and in the prior report suggest that inhibits transcription of the c-myc gene with a time course 1,25-(OH)2D3regulation of c-myc RNA levels and transcripthat could account for the decrease in c-myc mRNA levels. tion rate precedes its effect on DNA synthesis. The study Whether this effect is directly mediated by 1,25-(OH)2D3in reported here yields information that suggests direct expericombination with its receptor or results from secondary events mentation to determine if the1,25-(OH)2D3 receptor acts triggered by 1,25-(OH)2D3 treatment has notyet been estab- directly on DNA regions adjacent to promoter regions on the lished. It also remains possible that post-transcriptional pro- c-myc gene. Furthermore, studies to establish an invitro cessing could contribute in part to the in vivo regulation of transcription system to characterize the mechanism by which steady-state c-myc mRNA levels. the receptor interacts with the nucleus and DNA can now be The c-myc gene codes for a 60,000-dalton DNA binding initiated with c-myc as a probe for gene expression. protein localized in nuclei of all cells capable of cell division (17, 18, 32, 33). A t present, the exact function of the c-myc Acknowledgments-We thank Dr. Joel M. Depper, Department of gene product is not known. However, a relationship of c-myc Internal Medicine, University of Michigan, for helpful discussions, protein with DNA synthesis and cell proliferation has been T. Eng Gan and Emery Bresnick for preliminary experiments, and supported by data from a number of laboratories (17, 18, 32, Linda Harbison for secretarial assistance. 33). Whether the level of c-myc gene product is causally REFERENCES related to changes in cell proliferation or results from these changes has not been fully clarified. Previous work has shown 1. Franceschi, R. T., Simpson, R. U., and DeLuca, H. F. (1981) that the decrease in c-myc mRNAlevelsin Daudi human Arch. Bwchem. Biophys. 2 1 0 , 1-17 lymphoblastoid cells treated with interferon is not associated 2. Haussler, M. R., and McCain, T.A. (1977) N. Engl. J. Med. 2 9 7 , 974-978; 1041-1050 with a decrease in c-myc transcription rate, butwas associated 3. Norman, A. W., Roth, J., and Orci,L. (1982) Endocr. Reu. 3, with a decreased mRNA half-life (20). Other studies demon331-365 strating a marked effect of the protein synthesis inhibitor 4. Simpson, R. U., and Weishaar, R. J. (1986) Bio-Essays 3,44-48 cycloheximide on c-myc mRNA levels havesupported a major 5. Simpson, R. U., Hamstra, A. H., Kendrick, N. C., and DeLuca, rolefor post-transcriptional regulation of gene expression H. F.(1983) Biochemistry 22,2586-2594

with 1,25-(OH)ZD3 for periods of 12, 15, 24, and 48 h resulted in inhibition of transcription of c-myc ranging from 40% of control at 12 h to 100% at 48 h.Fig. 3A also shows that transcription of @-actinis not affected by 1,25-(OH)& during the course of treatment. Fig. 3B graphically demonstrates that 1,25-(OH)zD3specifically decreased transcription of the cmyc gene early in the course of differentiation. Comparison of data in Fig. 3 with the data shown in Fig. 2 reveals that a temporal correlation exists between the decrease in steadystate levels of c-myc RNA and the decrease in transcription of the gene. We examined the relationship of [32P]RNA input with relative levels of c-myc and @-actin transcription aftertreatment with 1,25-(OH)2D3(50 nM). In Fig. 4A we show that: 1) signal intensity for c-myc or @-actinhybridizations is a function of RNA input; 2) the c-myc/@-actinratio (cpm/cpm) is approximately 3.9 for this experiment (c-myc/actin ratios ranged from 0.8 to 3.9 in separate experiments with cultures with various growth rates and cell densities);and3) the decrease in c-myc levels after treatment with 1,25-(OH)zD3 (50 nM) is evident at all [32P]RNA inputsfrom 2 to 20 X lo6 cpm/ml RNA. Data in Fig. 4B show that c-myc/actin ratios (cpm/cpm) obtained by [32P]RNAelution and scintillation counting decreased by 75 to 90% after 72 h of 1,25-(OH)& (50 nM) treatment. We also found that while 50 nM inhibits transcription of c-myc, 1 nM had no effect on c-myc/actin ratios (data not shown).

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