Promyelocytes Protein in the Phorbol Ester-tolerant

Absence of Phorbol Ester-induced Down-Regulation of myc Protein in the Phorbol Ester-tolerant Mutant of HL-60 Promyelocytes David Gailani, Frank J. Cadwell, Peter S. O'Donnell, et al. Cancer Res 1989;49:5329-5333.

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[CANCER RESEARCH 49, 5329-5333, October 1. 1989]

Absence of Phorbol Ester-induced Down-Regulation of myc Protein in the Phorbol Ester-tolerant Mutant of HL-60 Promyelocytes1 David Gailani,2-3 Frank J. Cadwell,2 Peter S. O'Donnell, Robert A. Hromas,2 4 and Donald E. Macfarlane5 Department of Internal Medicine, Iowa City Veterans Administration Medical Center and University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242

ABSTRACT

in myc expression which occurs when S cells differentiate in response to TPA, TPA has no such effect on PET cells. This provides further evidence that down-regulation of myc is a prerequisite for cellular differentiation.

The human promyelocytic leukemia cell line HL-60 has an amplified number of copies of the protooncogene c-myc. It is induced to differentiate by exposure to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have developed a mutant phorbol ester-tolerant (PET) line of HL-60 which undergoes a transient growth arrest but does not differ entiate when exposed to TPA (Macfarlane et al., Br. J. Macinalo!.. 68: 291-302,1988). The defect is not due to a general failure of TPA-induced phosphorylation. In this paper, we show that exposing phorbol estersensitive (S) HL-60 cells to TPA caused the disappearance of the c-myc protein antigen (detected on Western blots) in 4 h, whereas TPA had no effect on the c-myc protein content of PET cells. Dimethyl sulfoxide caused the rapid disappearance of the myc antigen in both cells. PET cells had slightly more copies of the c-myc gene detected on Southern blots than S cells, c-myc mRNA was equally unstable in both cells, as determined by Northern blots following actinomycin D. TPA induced the down-regulation of e-nnr mRNA in S cells to a greater extent than in PET cells. Dimethyl sulfoxide caused a rapid down-regulation of c-myc mRNA in both cell lines. This shows that PET cells have a defect in the mechanism by which protein kinase C regulates c-myc transcription. Our results provide further evidence that reduction in c-myc expression is necessary for differentiation to occur in HL-60 cells.

MATERIALS AND METHODS Cell Lines. The HL-60 S cells and PET cells were maintained in suspension culture in RPMI 1640 medium with 10% heat inactivated fetal calf serum, 100 Â¿ig/mlstreptomycin and 100 units/ml penicillin, at 37Â°Cin a 5% CO2 humid atmosphere. The development and selection of the PET cell is described elsewhere (4). This line has been maintained in continuous culture since its selection, as has the parent S cell line. Cells were counted in a hemocytometer chamber after 1:1 dilution with trypan blue, 0.2% (w/v) in PBS. Western Blots of S and PET Cells. Ten million S or PET cells were withdrawn from culture and centrifuged at 500 x g for 4 min. After washing twice with PBS, the cells were resuspended in 0.5 ml of Hanks' balanced salt solution and an equal volume of SDS sample buffer was added [250 miviTris-HCl, pH 6.8; 20% (w/v) glycerol; 4% (w/v) SDS; 5% (v/v) 2-mercaptoethanol; and 0.01% (w/v) bromophenol blue]. Samples were sheared by being passed twice through a 20-gauge needle and 3 times through a 25-gauge needle and were stored at -20Â°C.The proteins contained in 150 p\ of these extracts (equivalent to 1.5 x IO6

INTRODUCTION The HL-60 promyelocytic goes terminal when exposed

cell line was established from a patient with leukemia (1) and is of interest because it under differentiation to cells with monocytic features to the phorbol ester TPA6 (2) and dihydroxy-

vitamin D., or to cells with granulocytic features when exposed to DMSO or retinoic acid (1-3). We have developed a mutant clone of HL-60 cells which does not differentiate when exposed to TPA (4). This PET cell line is sensitive to the differentiating effects of DMSO, dihydroxyvitamin D Â¡,and retinoic acid, and it displays the same altera tions in protein phosphorylation when exposed to TPA as does the phorbol ester-sensitive cell lines of "normal" HL-60 from which it was derived. PET cells are hypotetraploid, and they have a marker chromosome (iso(lp)) not present in S cells or other HL-60 variants (4). In this paper we report data concerning the regulation of the cellular oncogene c-myc and the protein that it encodes in this pair of cell lines. We show that, in contrast to the rapid decrease Received 11/2/88; revised 6/5/89; accepted 6/29/89. The costs of publication of this article were defrayed in part by the payment 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. ' This work was supported by a Merit Review Grant from the Veterans Administration. 2 Recipients of NIH Training Grant HL 07344. 3 Present address: Washington University School of Medicine, Barnard Cancer Center, St. Louis, MO 63110. ' Present address: University of Washington School of Medicine, Division of Hematology-Oncology, Seattle, WA 98105. 5To whom requests for reprints should be addressed, at Dept. of Internal Medicine, Division of Hematology-Oncology, University of Iowa Hospitals and Clinics, Iowa City, IA 52242. 'The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; DMSO, dimethyl sulfoxide; PET, phorbol ester tolerant; S, phorbol ester sensitive.

cells) were resolved by electrophoresis in 12.5% acrylamide gels (5) and were transferred to polyvinylidene difluoride (Millipore, Bedford, MA) filters using a Janssen (Piscataway, NJ) semidry blotter and the Trisaminohexanoate discontinuous buffer system described by the manu facturer. The filters were blocked by soaking in 50% skim milk at 37Â°Cfor 1 h and then were washed 3 times with PBS-0.1% Tween (10 min each wash). Filters were then incubated at 37Â°Cfor l h with the anti-human c-myc antibody (see below), washed 3 times with PBS-Tween, and then incubated at room temperature for l h with 15 ml of PBS-Tween containing 10 nCi of 125I-labeled goat anti-mouse IgG. Filters were again washed 3 times with PBS-Tween and autoradiographed for 48 h with Kodak XAR-5 film. Monoclonal Anti-Human c-myc Antibody. The hybridoma cell line Myc 1-9E10, secreting anti-m^c antibody (6), was maintained under the same conditions as the HL-60 cells. After centrifugation, the culture supernatant was stored at â€”¿20Â°C. Immediately before use, 6 ml were diluted to 15 ml with PBS-Tween. Southern Blots of c-myc Genomic DNA. Cells (6 x IO7) were lysed with Sarkosyl, digested with proteinase K, and extracted with phenolchloroform. The nucleic acids in the aqueous phase were precipitated with ice-cold 70% ethanol. This precipitant was redissolved in 10 mM Tris-HCl-0.1 mivi EDTA (pH 7.4) and digested for 30 min with 100 Mg/ml RNase A at 37"C before being extracted again with phenolchloroform. The DNA in the aqueous phase was digested with the restriction endonucleases Â£coRI,Hindlll, Sac\, Xmnl, or Pstl, accord ing to the supplier's directions. The incubation mixture was extracted with phenol-chloroform and the digested DNA was precipitated with cold 70% ethanol. After resuspension, the concentration of DNA was determined by absorbance at 260 nm in 10 mM Tris, 0.1 mM EDTA, pH 7.4. Serial dilutions of the DNA sequences were then electrophoresed on an agarose gel (0.8% (w/v) agarose in 80 mM Tris phosphate pH 8.0 2 mM EDTA-0.5 /ig/ml ethidium bromide), and the DNA was blotted onto nitrocellulose (7). This blot was treated for l h at 65Â°Cin 75 ml of 1%, w/v, polyvinylpyrrolidone, bovine serum albumin (lOx Denhardt's

5329


1% (w/v) Ficoll, 1% (w/v) solution), containing 0.2%

REGULATION OF myc IN PET HL-60

H97.4

66.2

Control 42.7

TPA

Fig. 1. Influence of differentiating agents on c-myc protein. S cells (left) or PET cells (right) were incubated without addition or with TPA (800 HM), DMSO (1.2%), dihydroxyvitamin I), (50 n\i), or retinoic acid (1 (.\i) (lower). At the times indicated (in h), samples of the cells were withdrawn and their content of c-myc protein was detected by Western blot as described in "Materials and Methods." Parts of the autoradiographs are shown. The migration of molecular size markers (in kDa) is indicated.

DMSO

vit D3

RA 0 2 46810

0 2 4 6 8 10

activity > IO8 dpm/jjg) (8), were added. The blots were allowed to hybridize for 16 h at 65Â°Cand then were washed 3 times for 15 min each with a solution of 300 HIMNaCl, 30 mM Na, citrate, 0.1% SDS, at room temperature, and then 3 times for 15 min each with a solution of 15 mM NaCl, 1.5 mM Na3 citrate, 0.1% SDS, at 65Â°C.The blots were dried and autoradiographed with Kodak XAR-5 film at â€”¿70Â°C,

6.0

with intensifier screens, for 5 days. Northern Blots. Samples containing 24 x 10'' cells were centrifuged

0

1

2

3

4

DAYS IN CULTURE

Fig. 2. Influence of TPA on the proliferation of PET cells. Culture medium was seeded with PET cells in the presence of 32 nM TPA (D) or in its absence (O). At times thereafter, samples were withdrawn and the concentration of cells was determined. Note that TPA causes a delay in the growth of the PET cells for about 1 day. The TPA-treated PET cells also exhaust the medium more rapidly, as evidenced by an early change in its color and a lower final cell count.

(w/v) SDS and 0.2 mg/ml salmon sperm DNA. After 1 h, 100 ng of 32P-nick-translated c-myc probe, prepared from a 1.3-kilobase ClalÂ£coRIfragment of the third exon of the human c-myc gene (specific 5330

at 500 x g for 5 min, washed twice with RPMI 1640 medium, and then dissolved in 2.5 ml of 4 M guanidinium isothiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M 2-mercaptoethanol, 0.5% Sarkosyl. One g CsCl was added and then the extract was layered onto 5.7 M CsCl, 0.1 M EDTA (pH 7.5), and centrifuged for 14 h at 35,000 rpm in a Beckman SW50.1 rotor (7). The RNA pellet was washed with 70% 2-propanol30% 0.2 M sodium acetate, dissolved in 0.1% (w/v) SDS-5 mM EDTA10 mM Tris buffer (pH 7.4), and precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.2 volumes of ethanol. The concentration of the RNA was determined spectrophotometrically at 260 nm. TwelveMgsamples were electrophoresed in a 1.5% agarose-6.7% formaldehyde gel in 3-[A/-morpholino]ethanesulfonic acid buffer, at 15 V/cm, for 4 h and then capillary-blotted onto Nytran paper (7). Blots were dried, hybridized with a human c-myc exon III probe (Oncor), and autoradi ographed. The blot was melted then reprobed with an actin probe



PET - cells

S - cells

PET Cells

S Cells

Kb I

1 =>

-12.8kb

PET Cells

S Cells

B

o o

LU

1 II -21 kb

-5.1 kb -4.9kb -4.2 kb -3.5 kb -2.0kb

Fig. 4. Genomic c-myc. A, semiquantitative Southern blots of DNA digested with EcoRl and probed with an EcoR\-Clal fragment from the third exon in cmyc. Serial dilutions of digests from PET cells and S cells are compared with a digest of human placenta DNA. The undiluted lanes contain 15 ng of DNA. Note that PET cell DNA is slightly enriched for c-myc, compared with S-cell DNA, and that both are markedly enriched compared with placenta! DNA. B, structure of c-myc gene. DNA from PET cells and S cells were digested with the restriction endonucleases indicated and electrophoresed. Blots were probed as above. The faint band at 4.9 kilobases in the PET cell Sad digest was not reproducible and is thought to be due to incomplete digestion.

HL-60 0

0124

1

30

60 120

15

30

60

120

2 4

Fig. 3. Influence of TPA and DMSO on c-myc RNA. S cells and PET cells were incubated with or without TPA (800 HM) or DMSO (1.2%) for the period indicated (in h). Cellular RNA was extracted, and mRNA for c-myc and actin was detected by Northern blot, as described in "Materials and Methods." Parts of the resulting autoradiographs are shown. The migration of RNA size markers is indicated.

(Oncor). The filters were autoradiographed for 12-36h.

15

PET

C-MYC

with Kodak XAR-5 film

ACTIN

RESULTS We determined the effect of inducers of differentiation on the amount of c-myc protein in S cells and PET cells by extracting them with SDS and performing Western blots on the extracts, using an anti-myc monoclonal antibody. The re sults (Fig. 1) show that TPA induces the rapid disappearance of the c-myc antigen from S cells, so that the signal in the Western blot becomes almost undetectable after 4 h of expo sure. In marked contrast, TPA caused no change in the amount of c-myc protein found in PET cells. In both S cells and PET cells, the level of c-myc decreased when the growth medium became exhausted and cellular proliferation ceased (data not shown). The effect of DMSO was similar to TPA, except that the PET cells and S cells were equally affected. Four h after the addition of DMSO, the antigen was barely detectable in either cell line. After prolonged incubation with retinoic acid or dihydroxy-vitamin D3, the c-myc mRNA of HL-60 cells is down-

Fig. 5. Stability of c-myc mRNA. PET cells and S cells were incubated with 5 fig of actinomycin D for the times indicated (min) before centrifugation and dissolution in guanidinium isothiocyanate, as described in "Materials and Meth ods" for the extraction of RNA. After electrophoresis. Northern blots were hybridized with c-myc (top) and a-actin (bottom) probes. Note that in both PET cells and S cells c-myc mRNA decays rapidly.

regulated (3). Our strains of HL-60 are less sensitive to these inducers than the original cell line is reported to be. The addition of dihydroxy-vitamin D3 to PET cells or S cells did not decrease the antigen in the period of our observations; indeed, there seemed to be some increase, although whether this increase was greater than that occurring in the control cells was not established. Retinoic acid did not induce a measurable change in antigen level in either cell line. The absence of a decrease in myc protein level induced by TPA in PET cells is not consistent with the notion that myc controls cell prolifera-

5331


REGULATION

OF myc IN PET HL-60

tion, because TPA induces a transient arrest in PET cell growth (Fig. 2). We examined the cellular content of c-myc mRNA by extract ing the cells with guanidinium isothiocyanate, isolating the whole-cell RNA by centrifugation, and hybridizing the North ern blots with radiolabeled sequences from the third exon of cmyc and then from the actin gene. This semiquantitative tech nique shows that both TPA and DMSO induce reduction in cmyc message in the S cells (Fig. 3). DMSO decreased the level in the PET cells. TPA induced a lesser degree of down-regula tion of c-myc mRNA in PET cells than S cells. Neither retinoic acid nor dihydroxy-vitamin Dj produced a consistent change in c-myc mRNA levels during 10 h of incu bation with S cells or PET cells (data not shown). We compared the copy number of the c-myc genes of S cells and PET cells by semiquantitative Southern blots of restriction endonuclease digests of genomic DNA. The blots were hybrid ized with an EcoRl-Clal fragment of the third exon of human c-myc. When expressed as a fraction of their DNA content, the PET cell is slightly enriched for c-myc sequences compared with the S cell (Fig. 4A). PET cells (being hypotetraploid) have nearly twice as much DNA/cell as do S cells, so that they have slightly more than twice as many copies of myc genes as S cells, expressed on a per cell basis. Restriction digestion with several endonucleases did not reveal any gross deletions, re arrangements, or insertions in the myc genes of PET cells (Fig. 4Ã„). In order to determine whether the persistence of the myc protein in PET cells was due to a stabilization of its mRNA, we treated S cells and PET cells with actinomycin D and estimated their content of c-myc mRNA at intervals thereafter by Northern blots (Fig. 5). This experiment shows that the ratio of c-myc mRNA to a-actin mRNA declines rapidly in both cell types, so that c-myc mRNA is barely detectable in either cell 1 h after RNA synthesis is inhibited.

occur. Transfection of U937 human monocytoid cells with a constitutively expressed \-myc gene prevents the induction of differentiation by DMSO (18), and suppressing the synthesis of the myc protein by exposing HL-60 cells to an unti-myc antisense oligonucleotide induces differentiation (19, 20). The myc gene encodes a nuclear protein whose biochemical function is disputed (21). We developed the PET cell clone in order to facilitate the investigation of the pharmacological coupling between the pro tein phosphorylation induced by phorbol esters and the mech anism of differentiation. The data we report here show that the PET cell differs markedly from the S cell in its regulation of cmyc. When S cells are exposed to TPA, their content of c-myc protein and c-myc mRNA decreases markedly within 4 h, whereas no such fall occurs when PET cells are exposed to TPA. We found that PET cells have slightly more copies of the cmyc gene than S cells, but this difference seems insufficient to account for the maintenance of myc protein in the PET cells. The cellular mRNA encoding myc is not more stable in PET cells than in S cells. We generated the PET cell line by chemical mutation and clonal selection. The phenotypic response of PET cells to TPA is strikingly similar to that of U937 cells transfected with constitutively expressed v-myc (18); in both cases, TPA induces a pause in proliferation for 1 day or so without differentiation to a fully adherent phenotype, followed by a resumption of normal growth. It is possible that the lesion responsible for the altered response of PET cells to TPA lies in the mechanism by which TPA down-regulates myc transcription. This conclusion gives further support for the necessity of down-regulation of myc expression for hematopoietic cells to express a differen tiated phenotype. ACKNOWLEDGMENTS We thank Dr. Gordon Cinder for useful discussions, David Marchant for technical assistance, and Judy Schroeder for typing the manuscript.

DISCUSSION The mechanism by which phorbol esters induce differentia tion is not known, but it presumably involves the activation of some genes and the reciprocal inactivation of others. Phorbol esters bind to protein kinase C and induce the phosphorylation of a number of proteins (9, 10). Several genes which are regu lated by exposure to phorbol esters have regulatory elements which bind transactivating proteins (11, 12). The avian myelocytomatosis virus is an acutely transforming retrovirus containing the owc-gene v-myc. The cellular homo logue of this gene, c-myc, is found to be translocated in Burkitt's lymphoma and murine plasmacytomas (13) and is amplified in HL-60. Mitogens induce the rapid accumulation of c-myc mRNA in a variety of quiescent cells, and when proliferating cells are induced to differentiate their content of c-myc mRNA falls. In HL-60 cells, this fall is due in part to the premature termination of RNA transcription before the coding region of the gene is reached, producing an unstable and untranslatable RNA species (14-16). As a result, little mature mRNA is synthesized and, since c-myc mRNA is degraded rapidly in HL60 (17), this interruption in synthesis has an early impact on the cellular content of c-myc mRNA. There is evidence to suggest that a reduction in cellular content of the c-myc protein is not merely one of many changes that result from cellular differentiation; rather, this reduction seems to be both sufficient and necessary for differentiation to

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to phorbol esters. Nature (Lond.), 329: 648-651, 1987. 12. Lee, W., Mitchell, P., and Tijan, R. Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell, 49: 741-752, 1987. 13. Dalia-Pavera. R.. Lombardi, L., Pelicci, P. G., Lanfrancone. L., Cesarman, E., and Neri, A. Mechanism of activation and biological role of the c-myc oncogene in B-cell lymphomagenesis. Ann. NY Acad. Sci., Sii: 207-218, 1987. 14. Bentley. D. L., and Groudine, M. A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells. Nature (Lond.), 321: 702-706, 1986. 15. Bentley, D. L., and Groudine, M. Sequence requirements for premature termination of transcription in the human c-myc gene. Cell, 53: 245-256, 1988. 16. Eick, D., and Bornkamm, G. W. Transcriptional arrest within the first exon is a fast control mechanism in c-myc gene expression. Nucleic Acid Res., 14: 8331-8346, 1986. 17. Dani. O. Blanchard, J. M.. Piechaczyk, M., El Sabouty, S., Marty, L., and

Jeanteur, P. H. Extreme instability of myc mRNA in normal and transformed human cells. Proc. Nati. Acad. Sci. USA, */: 7046-7050, 1984. 18. Larsson, L-G., Ivhed, I., Gidlund. M.. Pettersson, U., Vennstrom, B., and Nilsson, K. Phorbol ester-induced terminal differentiation is inhibited in human U-937 monoblastic cells expressing a v-myc oncogene. Proc. Nati. Acad. Sci. USA, 85: 2638-2642, 1988. 19. Wickstrom, E. L., Bacon, T. A., Gonzalez, A., Freeman, D. L., Lyman, G. H., and Wickstrom, E. Human promyelocytic leukemia HL-60 cell prolifer ation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide targeted against c-myc mRNA. Proc. Nati. Acad. Sci. USA, 85: 1028-1032, 1988. 20. Holt, J. T., Redner, R. L., and Nienhuis, A. W. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol. Cell Biol., 8: 963-973, 1988. 21. Gutierrez, C, Guo, Z-S., Burhans, W., DePamphilis, M. L., Farrell-Towt, J., and Ju, G. Is c-myc protein directly involved in DNA replication? Science (Wash. DC), 240: 1202-1203, 1988.

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