Vol.
167,
March
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16, 1990
Pages
Inducible
Shunji
Fourth
Gene Expression
Takahashi,
of Activin A/Erythroid in HL-60 Cells
Differentiation
654-658
Factor
Takayuki Yamas ita I, Yuzuru Eta*, Hiroshiro Kaoru Miyamoto 1 and Etsuro Ogata
Shibai*,
Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan * Central Research Laboratories, Ajinomoto Co. , Inc. , Kawasaki- ku , Kawasaki 210, Japan
# Department Received
January
of Obstetrics and Gynecology, Gunma University Medicine, Maebashi 371, Japan 25,
School of
1990
Treatment of HL-60 cells with 12-0-tetradecanoyl-phorbol13-acetate (TPA) for 48 h induced expression of mRNA of PA chain of activin A/erythroid differentiation factor. Under the same condition, interferon-y caused a slight increase in PA chain mRNA, whereas la, 25dihydroxyvitamin DB , dimethylsulfoxide and all-tram-retinoic acid failed to induce this mRNA in HL-60 cells. Furthermore, 4 h- treatment with TPA or lipopolysaccharide (LPS) induced a marked increase in PA chain mRNA levels in interferon-r-pretreated HL-60 cells. In the cells pretreated with la, 25-dihydroxyvitamin D 3, TPA and LPS induced as little increase in IA chain mRNA as in the control cells. Neither LYnor /3B chain mRNA was detected in any sample. These results indicate that interferon-y has a priming effect on the activation of activin A/erythroid differentiation factor gene by TPA or LPS in HL-60 cells. 0 1990 Academic mess,
Inc.
Activins and inhibins, stimulate
and inhibit
which were first purified from porcine ovarian fluid, respectively
secretion of follicle
(for a review, see Ref. 1) subunits,
Inhibins
common a and ,6A or /3B chain; activin
a heterodimer
of BA chain and /3B chain (activin
factor that induces erythroid supernatant designated
stimulating
differentiation
of 12-0-tetradecanoyl it as erythroid
hormone from cultured pituitary
are disulfide-bonded
dimer proteins
is a homodimer
differentiation
AB) . Independently,
factor
(EDF)
(2).
not only induces hemoglobin
cells but also enhances the growth 1
cells of two A) or
Eto et al. purified
Subsequently,
THP-1 cells and it was clarified
Other groups have reported
that
synthesis in K-562 cells, human erythroleukemia
and differentiation
of normal erythroid
and pluripotent
To whom correspondence should be addressed at Fourth Department of Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan.
Abbreviations: TPA, 12-0-tetradecanoyl-phorbol 13-acetate;EDF, erythroid differentiation factor; 1, 25(OH)2D3, la, 25-dihydroxyvitamin DB; IFN, interferon; DMSO, dimethylsulfoxide; LPS, lipopolysaccharide; NBT, nitroblue tetrazolium. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
654
a
cells from the culture
(TPA)-treated
that activin A and EDF are encoded by the same gene (3). activin A/EDF
of IA chain (activin
of murine erythroleukemia
phorbol13-acetate
constituted
Vol.
167,
No.
BIOCHEMICAL
2, 1990
hematopoietic
AND
precursor cells (4-6) . In addition,
in the rat bone marrow as a regulator
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
gene expression of activin A/EDF was detected
(7) . These observations
strongly
suggest that activin A/EDF
acts
Since THP-1 cell line is a monocytic leukemia
of hematopoiesis in the bone marrow.
cell line established from a patient with acute monocytic leukemia (8)) and is reported to produce interleukin
1 (9), activin A/EDF
However,
:little is known about the regulation
may be produced in cells of monocyte/macrophage
HL-60 cells are human promyelocytic macrophage-like see Ref. 10).
cells or granulocyte-like
all-trans-retinoic Furthermore,
Da (1, 25(OH)2D3)
of these cytokines.
A/EDF
differentiation
(IFN-7) (DMSO) ,
in these cells.
1 are known to be produced in HL-60 cells system to study regulation
In this study, we have investigated
of activin
and interferon-r
cells, whereas dimethylsulfoxide
E induce granulocytic
several cytokines such as interleukin
(11) . Thu,s, this cell line is a useful experimental expression
into monocyte/
cells in response to various agonists (for a review,
into monocyte/macrophage-like acid and prostaglandin
lineage.
of this factor in these cells.
leukemia cells and differentiate
TPA, la, 25-dihydroxyvitamin
induce differentiation
of production
the effects of various
of production agents on gene
in HL-60 cells. Materials and Methods
Chemicals TPA, all-trans-retinoic acid and lipopolysaccharide (LPS) (Escherichia coli, serotype 026: B6) were obtained from Sigma. 1, 25 (OH) 2D3 and purified, recombinant human IFN-r were kindly supplied from Chugai-Seiyaku Co. and Shionogi-Seiyaku Co., respectively. Other chemicals were obtained from commercial sources. Cell Culture ‘RIP-1 cells (8) were kindly supplied from Dr. S. Tsuchiya (Tohoku University). THP-1 and HL-60 cells were cultured in 5 ml RPM1 1640 medium supplemented with 10 %fetal calf serum under the humidified atmosphere of 5 %COz for 3-5 days at an initial cell density of 1 X lo5 cells/ml. For experiments, the cells were seeded at 5 X lo5 cells/ml in the serum-free medium with various stimulants, and cultured for indicated time. Then the cells were processed to RNA analysis. Monocytic or granulocytic differentiation was evaluated by the percentages of morphologically maturated cells in May-Giemsa-stained cell preparations, nitroblue tetrazolium (NBT) test-positive cells (12), and nonspecific esterase-positive cells. RNA blot analysis cDNAs of human @A, LYand ,!?Bchains were previously described (3, 13, 14) . Human ,&actin genomic DNA was kindly supplied from Dr. M. Yoshida (Cancer Institute The probes were labeled with [3ZP]dCTP by Nick translation kit (Takara Shuzo of Japan), Co. , Tokyo, Japan) - Total cellular RNA was extracted from the cells by guanidium thiocyanate method (1’5) . Poly(A)+RNA was selected by oligo(dT) -cellulose column chromatography (16). Ten micrograms of total or poly(A)+RNA were separated on 1 %formaldehyde/agarose gel by electrophoresis and blotted onto nylon membrane filters (Pall Ultrafine Filtration Corp. , Glen Cove, N . Y. ) . The filters were hybridized with 32P-labeled probes at 42’c in 5 x SSC (SSC : 0.15 M NaCl/0.015 M sodium citrate), 50 %formamide, 40,ug/ml poly(A) and 0.1% sodium dodecyl sulfate overnight and washed at 60C in 0.1 x SSC and 0.1% sodium dodecyl sulfate. Autoradiography was performed with intensifying screen at -80°C for 24 hours or longer. Invariability of the amounts of RNA loaded in each lane was checked by rehybridisation with ,9-actin probe. Results and Discussion It was reported that the bioactivity of TPA-treated
of activin A/EDF was detected in the supernatant
HL-60 cells as judged by erythrodifferentiation
cells (17) . To confirm
this observation,
of murine erythroleukemia
we examined whether TPA treatment of HL-60 cells could induce an increase of activin A/EDF mRNA. THP- 1 and HL-60 cells were cultured for 655
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
48 hours with 10 nM TPA and expression of /3A chain mRNA was evaluated by northern
blot
analysis (Fig. 1). In TPA-stimulated
THP-1 cells, we could detect four /?A chain mRNAs (about
6.Okb,
andthe2.8kbmRNAwasamainbandconsistentwith
4.0kb,
2.8kband1.7kb),
the observation in a preceding report (3) . Also in TPA-stimulated
HL-60 cells, ,!?Achain mRNAs
could be detected, but main mRNAs were larger (about 6.0 kb and 4.5 kb) . Molecular for multiplicities of multiple
of @A chain mRNA is unknown.
lengths due to alternative
basis
However, it is possible that there are mRNAs
splicing or use of different
polyadenilation
sites, since
7.2kb and 4.5kb /3A chain mRNAs are reported also in porcine ovary and testis (18) . In the same samples, confirm
neither rx nor ,JB chain mRNA was detected (data not shown)
that activin A/EDF
is produced
in TPA-treated
Since TPA is known to induce HL-60 cell differentiation cells (19), we examined the effect of various differentiation
. Our results
HL-60 cells. into monocyte/macrophage-like inducers on activin A/EDF
mRNA
levels in HL-60 cells. HL-60 cells were cultured for 48 hours in the presence of TPA, IFN-r 1, 25(OH)eD3, evaluated
DMSO,
by northern
or all-trans-retinoic blot analysis
mRNAs in HL-60 cells. 1,
25(OH)2D3,
DMSO,
(Fig.
acid.
Then expression
,
of BA chain mRNA was
2) . IFN-7 induced a slight increase of ,9A chain
We could not detect BA chain mRNA in the cells treated by or all-trans-retinoic
acid. In the same samples, neither cynor BB chain
mRNA was detected (data not shown) . With any inducer, most of the cells morphologically maturated. DMSO-
The percentages
, and all-trans-retinoic
respectively.
of NBT test-positive
cells in TPA-,
1 ,25(OH)zD3-,
acid- treated cells were about lo%, 90%) 80%, 55%, and 45%,
The percentages of nonspecific esterase-positive
(50-60%) among TPA- , IFN-r
IFN-r-,
cells did not differ significantly
- , and 1,25 (OH) 2D3 - treated cells.
TPA and LPS are known to activate see Ref. 20) . In the last experiment,
a mature monocyte/macrophage(for
a review,
we examined the effects of these agents on expression
12345 1234 28 S-
18 S -
02
01 Figure 1. Expression of was applied per lane. Lane 1, total RNA of of HL-60 cells; lane 4, shown on the left.
DA chain mRNA in TPA-treated HL-6ocells. 10% total or The filter was hybridized with the 32P-labeled ,9A chain THP-1 cells; lane 2, poly (A)+RNA of THP-1 cells; lane poly(A)+RNA of HL-60 cells. The locations of 28s and
poly(A)+RNA cDNA probe. 3, total RNA 18s rRNA are
Figure 2. Effects of various differentiation inducers on BA chain mRNA expression in HL-60 cells. Total RNA (lo& was isolated from HL-60 cells cultured with various differentiation inducers for 48 hours. Lane 1, 10 nM TPA; lane 2, 1000 U/ml IFN-I ; lane 3, 10 nM 1, 25(OH)eDa; lane 4, 1.3 9: DMSO; lane 5, 1 PM all-tyans-retinoic acid. The filter was hybridized with the a2P-labeled ,4A chain cDNA probe. The locations of 28s and 18s rRNA are shown on the left.
656
Vol. 167, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12345678
28 s18 S-
Figure 3. Effects of TPA and LPS on @A chain mRNA expression in HL-60 cells differentiated -by IFN-r or 1, 25(OH)zD3. Total RNA (10,ag) was isolated from HL-60 cells pretreated with 1000 U/ml IFN-r or 10 nM 1, 25(OH)zD3 for 43 hours and sequentially stimulated with 10 nM TPA or 1 ,ug/ml LPS for 4 hours. Lane 1, with no agonists for 48 hours and TPA for 4 hours; lane 2, TPA alone for 48 hours; lane 3, IFN-r for 48 hours and TPA for 4 hours; lane 4, 1, 25(OH)pDa for 48 hours and TPA for 4 hours; lane 5, TPA alone for 48 hours; lane 6, with no agonists for 48 hours and LPS for 4 hours; lane 7, IFN-r for 48 hours and LPS 4 hours; lane 8, 1, 25(OH)*D3 for 48 hours and LPS for 4 hours. The filter was hybridized with the 32P-latleled /3A chain cDNA probe. The locations of 28s and 18s rRNA are shown on the left.
of activin A/EDF
gene in HL-60 cells differentiated
by IFN-r
or 1, 25(OH)sDs.
HL-60 cells
were culturled for 43 hours with IFN- y or 1, 25 (OH) 2D3 and then TPA (10 nM) or LPS (1 ,ug/ml) was added. pretreated
After 4 hours, expression of /3A chain mRNA was evaluated with IFN-r , TPA and LPS markedly
in the cells pretreated
(Fig. 3) . In the cells
increased BA chain mRNA levels. In contrast,
with 1, 25 (OH) 2D3, these agents induced as little increase in PA chain
mRNA as in the control cells. Again, in the same samples, LYor /?B chain mRNA was not detected (data not shown). An interesting on activin
A/EDF
finding
of our results is that IFN-7 exhibited
with TPA or LPS. This finding suggests that IFN-r
activate th:i gene by different mechanisms. of HL-60 cells (21).
However, monocytic
effect on the activin A/EDF
differentiation
gene activation,
may be insufficient
for this priming
since 1, 25 (OH) 2D3, which induced nonspecific
cells to a similar extent, had no priming effect. Although a reason for different
HL-60 cell differentiation
(22).
1 in peripheral mechanisms
one explanation
into monocyte/macrophage-like
monocyte/macrophage molecular
and TPA or LPS
IFN-7 is known to induce monocytic differentiation
effects of I:FN-7 and 1, .25(OH) 2D3 is unknown,
interleukin
effect
gene expression in HL-60 cells as judged by a rapid increase of this mRNA
by sequent:ial treatment
e&erase-polsitive
a marked priming
In fact, a similar
blood monocytes
for regulation
priming
was reported
of activin
A/EDF
is that IFN-r
not only induces
cells but also activates a mature effect of IFN-r
on expression
of
(23) . We are currently
studying
gene expression by IFN-r
and TPA
or LPS in HL-60 cells. Acknowledgments We would like to thank Dr. Shigeru Tsuchiya (Tohoku University) for allowing us to use ‘HIP-1 cells. This work was supported in part by grants from the Ministry of Education, Science and Culture. 657
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
References 1. Ying, S. (1988) Endocrine Reviews, 9, 267-293. 2. Eta, Y. , Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y. and Shibai, H. (1987) Biochem. Biophys. Res. Commun. , 142, 1095-1103. 3. Murata v M., Eta, Y. , Shibai, H., Sakai, M. and Muramatsu, M. (1988) proc. NatI. Acad. Sci. USA, 85, 2434-2438. 4. Yu, J.9 Shao, L., Lemas, V., Yu, A.L., Vaughan, J., Rivier, J. andVale, W. (1987) Nature, 330, 765-767. 5. Broxymeyer, H.E., Lu, L., Cooper, S., Schwall, R.H. , Mason, A. J. and Nikolics, K. (1988) F’roc. Natl. Acad. Sci. USA, 85, 9052-9056. 6. Yu, J. q Shao, L., Vaughan, J., Vale, W. and Yu, A.L. (1989) Blood, 73, 952-960. 7. Meunier, H. , Rivier, C. , Evans, R.M. and Vale, W. (1988) Proc. NatI. Acad. Sci. USA, 85, 247-251. 8. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y. , Konno, T. and Tada, K. (1980) Int. J. Cancer, 26, 171-176. 9. Krakauer , ‘I’. and Oppenheim , J . J. (1983) Cell. Immunol . , 80, 223-229. 10. Collins, S.J. (1987) Blood, 70, 1233-1244. 11. Spear, G. T. , Paulnock, D.M., Helgeson, D.O. and Borden, E.C. (1988) Cancer Res., 48, 1740-1744. 12. CoIlins, S. J. , Ruscetti, F. W. ,Gallagher, R.E. and Gallo, R.C. (1979) J. Exp. Med. 149,969-974. 13. Fukuda, M., Miyamoto, K., Hasegawa, Y., Nomura, M. , Igarashi, M., Kangawa, K. and Matsuo , H. (1986) Mol. Cell. Endocrinol. , 44, 55-60 14. Mason, A.J., Niall, H.D. and Seeburg, P.H. (1986) B&hem. Biophys. I&s. Comun., 135,957-964. 15. Chirgwin, J. , Przybyla, A. E. , MacDonald, R. J. and Rutter, W. J. (1979) Bioc&m&.ry, 18,5298-5299.
16. Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. USA, 69, 1408-1412. 17. Tsuji, T. I Eta, Y. I Takano, S. , Takezawa, M. , Yokogawa, Y. and Shibai, H. (1987) Biotechnology and Bioengineering , 31, 675-681. 18. Lee, W. , Mason, A. J. , Schwall, R. , Szonyi, E. and Mather, J. P. (1989) Science, 243, 396-398. 19. Rovera, G. , O’Brien, T.G. and Diamond, L. (1979) Science, 204, 868-870. 20. Adams, D.O. andHamilton, T.A. (1984) Ann. Rev. Immunol., 2, 283-318. 21. Ball, E. D. I Guyre, P. , Shen, L. , Glynn, J. M. , Maliszewski, C. R. , Baker, p. E and Fanger, M. W. (1984) J. Clin. Invest. , 73, 1072-1077. 22. Nathan, C.F. , Prendergast, T. J. , Wiebe, M. E., Stanley, E.R. , Platzer, E. , Remold, H.G., Welte, K., Rubin, B.Y. andMurray, H.W. (1984) J. Exp. Med., 160, 690-605. 23. Arenzana-Seisdedos , F . , Virelizier , J. L . and Fiers , W . (1985) J. ImmunoI . , 134, 2444-2448.
658
167,
March
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16, 1990
Pages
Inducible
Shunji
Fourth
Gene Expression
Takahashi,
of Activin A/Erythroid in HL-60 Cells
Differentiation
654-658
Factor
Takayuki Yamas ita I, Yuzuru Eta*, Hiroshiro Kaoru Miyamoto 1 and Etsuro Ogata
Shibai*,
Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan * Central Research Laboratories, Ajinomoto Co. , Inc. , Kawasaki- ku , Kawasaki 210, Japan
# Department Received
January
of Obstetrics and Gynecology, Gunma University Medicine, Maebashi 371, Japan 25,
School of
1990
Treatment of HL-60 cells with 12-0-tetradecanoyl-phorbol13-acetate (TPA) for 48 h induced expression of mRNA of PA chain of activin A/erythroid differentiation factor. Under the same condition, interferon-y caused a slight increase in PA chain mRNA, whereas la, 25dihydroxyvitamin DB , dimethylsulfoxide and all-tram-retinoic acid failed to induce this mRNA in HL-60 cells. Furthermore, 4 h- treatment with TPA or lipopolysaccharide (LPS) induced a marked increase in PA chain mRNA levels in interferon-r-pretreated HL-60 cells. In the cells pretreated with la, 25-dihydroxyvitamin D 3, TPA and LPS induced as little increase in IA chain mRNA as in the control cells. Neither LYnor /3B chain mRNA was detected in any sample. These results indicate that interferon-y has a priming effect on the activation of activin A/erythroid differentiation factor gene by TPA or LPS in HL-60 cells. 0 1990 Academic mess,
Inc.
Activins and inhibins, stimulate
and inhibit
which were first purified from porcine ovarian fluid, respectively
secretion of follicle
(for a review, see Ref. 1) subunits,
Inhibins
common a and ,6A or /3B chain; activin
a heterodimer
of BA chain and /3B chain (activin
factor that induces erythroid supernatant designated
stimulating
differentiation
of 12-0-tetradecanoyl it as erythroid
hormone from cultured pituitary
are disulfide-bonded
dimer proteins
is a homodimer
differentiation
AB) . Independently,
factor
(EDF)
(2).
not only induces hemoglobin
cells but also enhances the growth 1
cells of two A) or
Eto et al. purified
Subsequently,
THP-1 cells and it was clarified
Other groups have reported
that
synthesis in K-562 cells, human erythroleukemia
and differentiation
of normal erythroid
and pluripotent
To whom correspondence should be addressed at Fourth Department of Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan.
Abbreviations: TPA, 12-0-tetradecanoyl-phorbol 13-acetate;EDF, erythroid differentiation factor; 1, 25(OH)2D3, la, 25-dihydroxyvitamin DB; IFN, interferon; DMSO, dimethylsulfoxide; LPS, lipopolysaccharide; NBT, nitroblue tetrazolium. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
654
a
cells from the culture
(TPA)-treated
that activin A and EDF are encoded by the same gene (3). activin A/EDF
of IA chain (activin
of murine erythroleukemia
phorbol13-acetate
constituted
Vol.
167,
No.
BIOCHEMICAL
2, 1990
hematopoietic
AND
precursor cells (4-6) . In addition,
in the rat bone marrow as a regulator
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
gene expression of activin A/EDF was detected
(7) . These observations
strongly
suggest that activin A/EDF
acts
Since THP-1 cell line is a monocytic leukemia
of hematopoiesis in the bone marrow.
cell line established from a patient with acute monocytic leukemia (8)) and is reported to produce interleukin
1 (9), activin A/EDF
However,
:little is known about the regulation
may be produced in cells of monocyte/macrophage
HL-60 cells are human promyelocytic macrophage-like see Ref. 10).
cells or granulocyte-like
all-trans-retinoic Furthermore,
Da (1, 25(OH)2D3)
of these cytokines.
A/EDF
differentiation
(IFN-7) (DMSO) ,
in these cells.
1 are known to be produced in HL-60 cells system to study regulation
In this study, we have investigated
of activin
and interferon-r
cells, whereas dimethylsulfoxide
E induce granulocytic
several cytokines such as interleukin
(11) . Thu,s, this cell line is a useful experimental expression
into monocyte/
cells in response to various agonists (for a review,
into monocyte/macrophage-like acid and prostaglandin
lineage.
of this factor in these cells.
leukemia cells and differentiate
TPA, la, 25-dihydroxyvitamin
induce differentiation
of production
the effects of various
of production agents on gene
in HL-60 cells. Materials and Methods
Chemicals TPA, all-trans-retinoic acid and lipopolysaccharide (LPS) (Escherichia coli, serotype 026: B6) were obtained from Sigma. 1, 25 (OH) 2D3 and purified, recombinant human IFN-r were kindly supplied from Chugai-Seiyaku Co. and Shionogi-Seiyaku Co., respectively. Other chemicals were obtained from commercial sources. Cell Culture ‘RIP-1 cells (8) were kindly supplied from Dr. S. Tsuchiya (Tohoku University). THP-1 and HL-60 cells were cultured in 5 ml RPM1 1640 medium supplemented with 10 %fetal calf serum under the humidified atmosphere of 5 %COz for 3-5 days at an initial cell density of 1 X lo5 cells/ml. For experiments, the cells were seeded at 5 X lo5 cells/ml in the serum-free medium with various stimulants, and cultured for indicated time. Then the cells were processed to RNA analysis. Monocytic or granulocytic differentiation was evaluated by the percentages of morphologically maturated cells in May-Giemsa-stained cell preparations, nitroblue tetrazolium (NBT) test-positive cells (12), and nonspecific esterase-positive cells. RNA blot analysis cDNAs of human @A, LYand ,!?Bchains were previously described (3, 13, 14) . Human ,&actin genomic DNA was kindly supplied from Dr. M. Yoshida (Cancer Institute The probes were labeled with [3ZP]dCTP by Nick translation kit (Takara Shuzo of Japan), Co. , Tokyo, Japan) - Total cellular RNA was extracted from the cells by guanidium thiocyanate method (1’5) . Poly(A)+RNA was selected by oligo(dT) -cellulose column chromatography (16). Ten micrograms of total or poly(A)+RNA were separated on 1 %formaldehyde/agarose gel by electrophoresis and blotted onto nylon membrane filters (Pall Ultrafine Filtration Corp. , Glen Cove, N . Y. ) . The filters were hybridized with 32P-labeled probes at 42’c in 5 x SSC (SSC : 0.15 M NaCl/0.015 M sodium citrate), 50 %formamide, 40,ug/ml poly(A) and 0.1% sodium dodecyl sulfate overnight and washed at 60C in 0.1 x SSC and 0.1% sodium dodecyl sulfate. Autoradiography was performed with intensifying screen at -80°C for 24 hours or longer. Invariability of the amounts of RNA loaded in each lane was checked by rehybridisation with ,9-actin probe. Results and Discussion It was reported that the bioactivity of TPA-treated
of activin A/EDF was detected in the supernatant
HL-60 cells as judged by erythrodifferentiation
cells (17) . To confirm
this observation,
of murine erythroleukemia
we examined whether TPA treatment of HL-60 cells could induce an increase of activin A/EDF mRNA. THP- 1 and HL-60 cells were cultured for 655
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
48 hours with 10 nM TPA and expression of /3A chain mRNA was evaluated by northern
blot
analysis (Fig. 1). In TPA-stimulated
THP-1 cells, we could detect four /?A chain mRNAs (about
6.Okb,
andthe2.8kbmRNAwasamainbandconsistentwith
4.0kb,
2.8kband1.7kb),
the observation in a preceding report (3) . Also in TPA-stimulated
HL-60 cells, ,!?Achain mRNAs
could be detected, but main mRNAs were larger (about 6.0 kb and 4.5 kb) . Molecular for multiplicities of multiple
of @A chain mRNA is unknown.
lengths due to alternative
basis
However, it is possible that there are mRNAs
splicing or use of different
polyadenilation
sites, since
7.2kb and 4.5kb /3A chain mRNAs are reported also in porcine ovary and testis (18) . In the same samples, confirm
neither rx nor ,JB chain mRNA was detected (data not shown)
that activin A/EDF
is produced
in TPA-treated
Since TPA is known to induce HL-60 cell differentiation cells (19), we examined the effect of various differentiation
. Our results
HL-60 cells. into monocyte/macrophage-like inducers on activin A/EDF
mRNA
levels in HL-60 cells. HL-60 cells were cultured for 48 hours in the presence of TPA, IFN-r 1, 25(OH)eD3, evaluated
DMSO,
by northern
or all-trans-retinoic blot analysis
mRNAs in HL-60 cells. 1,
25(OH)2D3,
DMSO,
(Fig.
acid.
Then expression
,
of BA chain mRNA was
2) . IFN-7 induced a slight increase of ,9A chain
We could not detect BA chain mRNA in the cells treated by or all-trans-retinoic
acid. In the same samples, neither cynor BB chain
mRNA was detected (data not shown) . With any inducer, most of the cells morphologically maturated. DMSO-
The percentages
, and all-trans-retinoic
respectively.
of NBT test-positive
cells in TPA-,
1 ,25(OH)zD3-,
acid- treated cells were about lo%, 90%) 80%, 55%, and 45%,
The percentages of nonspecific esterase-positive
(50-60%) among TPA- , IFN-r
IFN-r-,
cells did not differ significantly
- , and 1,25 (OH) 2D3 - treated cells.
TPA and LPS are known to activate see Ref. 20) . In the last experiment,
a mature monocyte/macrophage(for
a review,
we examined the effects of these agents on expression
12345 1234 28 S-
18 S -
02
01 Figure 1. Expression of was applied per lane. Lane 1, total RNA of of HL-60 cells; lane 4, shown on the left.
DA chain mRNA in TPA-treated HL-6ocells. 10% total or The filter was hybridized with the 32P-labeled ,9A chain THP-1 cells; lane 2, poly (A)+RNA of THP-1 cells; lane poly(A)+RNA of HL-60 cells. The locations of 28s and
poly(A)+RNA cDNA probe. 3, total RNA 18s rRNA are
Figure 2. Effects of various differentiation inducers on BA chain mRNA expression in HL-60 cells. Total RNA (lo& was isolated from HL-60 cells cultured with various differentiation inducers for 48 hours. Lane 1, 10 nM TPA; lane 2, 1000 U/ml IFN-I ; lane 3, 10 nM 1, 25(OH)eDa; lane 4, 1.3 9: DMSO; lane 5, 1 PM all-tyans-retinoic acid. The filter was hybridized with the a2P-labeled ,4A chain cDNA probe. The locations of 28s and 18s rRNA are shown on the left.
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28 s18 S-
Figure 3. Effects of TPA and LPS on @A chain mRNA expression in HL-60 cells differentiated -by IFN-r or 1, 25(OH)zD3. Total RNA (10,ag) was isolated from HL-60 cells pretreated with 1000 U/ml IFN-r or 10 nM 1, 25(OH)zD3 for 43 hours and sequentially stimulated with 10 nM TPA or 1 ,ug/ml LPS for 4 hours. Lane 1, with no agonists for 48 hours and TPA for 4 hours; lane 2, TPA alone for 48 hours; lane 3, IFN-r for 48 hours and TPA for 4 hours; lane 4, 1, 25(OH)pDa for 48 hours and TPA for 4 hours; lane 5, TPA alone for 48 hours; lane 6, with no agonists for 48 hours and LPS for 4 hours; lane 7, IFN-r for 48 hours and LPS 4 hours; lane 8, 1, 25(OH)*D3 for 48 hours and LPS for 4 hours. The filter was hybridized with the 32P-latleled /3A chain cDNA probe. The locations of 28s and 18s rRNA are shown on the left.
of activin A/EDF
gene in HL-60 cells differentiated
by IFN-r
or 1, 25(OH)sDs.
HL-60 cells
were culturled for 43 hours with IFN- y or 1, 25 (OH) 2D3 and then TPA (10 nM) or LPS (1 ,ug/ml) was added. pretreated
After 4 hours, expression of /3A chain mRNA was evaluated with IFN-r , TPA and LPS markedly
in the cells pretreated
(Fig. 3) . In the cells
increased BA chain mRNA levels. In contrast,
with 1, 25 (OH) 2D3, these agents induced as little increase in PA chain
mRNA as in the control cells. Again, in the same samples, LYor /?B chain mRNA was not detected (data not shown). An interesting on activin
A/EDF
finding
of our results is that IFN-7 exhibited
with TPA or LPS. This finding suggests that IFN-r
activate th:i gene by different mechanisms. of HL-60 cells (21).
However, monocytic
effect on the activin A/EDF
differentiation
gene activation,
may be insufficient
for this priming
since 1, 25 (OH) 2D3, which induced nonspecific
cells to a similar extent, had no priming effect. Although a reason for different
HL-60 cell differentiation
(22).
1 in peripheral mechanisms
one explanation
into monocyte/macrophage-like
monocyte/macrophage molecular
and TPA or LPS
IFN-7 is known to induce monocytic differentiation
effects of I:FN-7 and 1, .25(OH) 2D3 is unknown,
interleukin
effect
gene expression in HL-60 cells as judged by a rapid increase of this mRNA
by sequent:ial treatment
e&erase-polsitive
a marked priming
In fact, a similar
blood monocytes
for regulation
priming
was reported
of activin
A/EDF
is that IFN-r
not only induces
cells but also activates a mature effect of IFN-r
on expression
of
(23) . We are currently
studying
gene expression by IFN-r
and TPA
or LPS in HL-60 cells. Acknowledgments We would like to thank Dr. Shigeru Tsuchiya (Tohoku University) for allowing us to use ‘HIP-1 cells. This work was supported in part by grants from the Ministry of Education, Science and Culture. 657
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