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Cooperative effects of tumor necrosis factor-α and 1,25-dihydroxyvitamin D3 on growth inhibition, differentiation, and c-myc reduction in human promyelocytic leukemia cell line HL-60
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages I151-1157
Vo1.152, No. 3,1988 May 16,1988
COOPERATIVE EFFECTS OF TUMOR NECROSIS FACTOR-~ AND 1,25-DIHYDROXYVITAMIN D 3 ON GROWTH INHIBITION,
DIFFERENTIATION, AND c-myc REDUCTION IN HUMAN
PROMYELOCYTIC LEUKEMIA CELL LINE HL-60*
Yuko Katakami §, Yoshimobu Nakao, Nobuyuki Kat~ami, Tamio Koizumi, Ryoichi Ogawa, Hiroki Yamada, Yoshimi Takai' and Takuo Fujita
Departments of M~dicin~ (Third D i v i s i o n ) and B i o c h e m i s t r y ~ K o ~ U n i v e r s i t y School of Medicine, Kob~ 650, japan Received March 14, 1988 SUMMARY: The simultaneous addition of recombinant human tumor necrosis factor(rTNF-~) and 1,25-dihydroxyvitamin D 3 (1,25-(OH)2D3) synergistically induced growth inhibition and differentiation of the human promyelocytic leukemia cell line HL-60. A significantly greater reduction in c-myc mRNA level was also observed after a 20-h combined treatment with rTNF-~ and 1,25-(OH)2D 3 than was observed following administration of either agent alone. These results suggest that rTNF-~ and 1,25-(OH)2D 3 induce HL-60 cell growth inhibition through different molecular mechanisms, and that their simultaneous administration could provide a new approach to the treatment of cancer. © z988Acaaemic Press,
Inc.
The monokine, TNF-~
, was originally identified by its cytostatic or
cytotoxic effect on some tumor cells (i-3). Due to its apparent tumor selectivity (1-3), intense interest concerning potential clinical application of TNF-~ has been stimulated.
However,
its cytostatic/cytotoxic activity
not all human tumors are sensitive to
(2,3).
Recent studies have shown that the combined treatment of rTNF-a with rlFN-T has a synergistic antiproliferative effect on certain tumor cell lines that are resistant or less sensitive to rTNF-~ (3,4). Synergism between rTNF-
* This investigation was supported in part by a Grant-in Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. § To whom correspondence should be addressed. Abbreviations used are: TNF-~, tumor necrosis factor-a; rTNF-~, recombinant human TNF-e; rIFN-y, recombinant human interferon-y; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; NBT, nitroblue tetrazolium; PBS, phosphate-buffered saline. 0006-291X/88 $1.50 1151
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and rlFN-y to induce differentiation of myeloid leukemia cell lines has also been reported (5).
It is therefore of interest to test for synergism between
rTNF-~ and other naturally occurring growth inhibitors.
One of such growth
inhibitors that has been shown to exert an antiproliferative effect on certain tumor cells is 1,25-(OH)2D 3 (6-11). The human promyelocytic leukemia cell line HL-60 responds to 1,25-(OH)2D 3 by ceasing division and acquiring some of the characteristics of mature myeloid cells, and these phenotypic changes are preceded by the reduction in the expression of the amplified protooncogene, c-myc (8, 11-13).
In the
present studies we have therefore examined the possible cooperative effects of rTNF-~ and 1,25-(OH)2D 3 on growth inhibition,
differentiation,
and reduction
of the level of c-~yc mRNA expression in HL-60 cells.
MATERIALS AND METHODS
~rmf Calf C a ~ - - T h e human promyelocytic leukemia cell line HL60 was kindly provided by Dr. R.C. Gallo (National Cancer Institute, Bethesda, MD). The cells were cultured at 37°C in RPMI-1640 medium (M.A. Bioproduct) supplemented with 25 m~ HEPES buffer, kanamycin (120 Bg/ml), and 10% heatinactivated fetal calf s e r u ~ i n a humidified atmosphere of 5% CO 2 in air. The cells were seeded at 1 x l O J cells per ml in culture flasks and incubated in the presence of various concentrations of rTNF-~ and 1,25-(OH)2D $. The latter was dissolved in ethanol at such a concentration that, when a p p h e d to test cultures, the final concentration of the solvent was less than 0.1%. A control culture medium containing 0.1% ethanol had no effect on HL-60 cell growth or differentiation. /leagea~ - - P u r i f i e d rTNF-~ derived from E s ~ s h g a cof2 (14) was a generous gift from A~ahi Chemical Industry Co., Ltd. It had a specific activity of 2.3 x i0 u units per mg of protein. 1,25-(OH)2D 3 was kindly provided by the Teijin Institute for Biomedical Research. NBT was purchased from Sigma. C a ~ G ~ o w ~ S i MaasaJuz~ar~!--Plastic-adherent cells were detached by gentle pipetting and cell number was determined with a hemocytometer. Viability was determined by the ability of cells to exclude trypan blue. Dif~gaan~££o~ ~ s a g - - D i f f e r e n t i a t i o n was assayed by the ability of cells to reduce NBT to formazan as described previously (ii). Me~sara~aa~ of c-~yc m/97VA--Cells were harvested and washed at 4°C with PBS. Total cellular RNA was extracted by the procedure of Chirgwin at ~ . (15), utilizinR CsCI gradient centrifugation. For Northern blot hybridization, total cellular RNA was denatured, electrophoresed (i0 Bg RNA/lane) in a 1% agarose slab gel, and was transferred to a nitrocellulose filter as described previously (Ii).~^ After baking, the filters were hybridized for 48 h at 42°C with a JzP-labeled DNA probe. The unbound fraction was then removed by extensive washing (ii). The filters were autoradiographed using intensifying screens at -80°C. For dot blot analysis, total cellular RNA samples were dotted onto nitrocellulose filters under a sl@~ vacuum. After baking, the filters were hybridized for 48 h at 42°C with a J~P-labeled DNA probe, and the unbound fraction was then removed by
1152
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
extensive washing. The filters were autoradiographed using intensifying screens for 24 h at -80°C, and the dots were excised and counted in a scintillation counter. Within the range of 1 to I0 ~g the amount of probe hybridized was proportional to the amount of RNA on the filter. The ~ v e l s c-myc mRNA are expressed as cpm per 4 ~g of total cellular RNA. The Plabeled DNA probe used was a nick-translated 5.5-kb Bam HI fragment, containing mouse c-myc gene segments that had been isolated from a mouse plasmacytoma SI07 bacteriophage library and subcloned into pBR322 (16).
of
RESULTS When HL-60 cells were cultured with rTNF-a alone for 4 days, cell growth was slightly inhibited in a dose-dependent manner, with the viable cell number being maximally decreased only to 85% of the control level at concentrations of more than I00 U/ml as shown in Fig. i.
1,25-(OH)2D 3 alone at a
concentration of I0 nM also slightly inhibited HL-60 cell growth and the viable cell number decreased only to 86%. added simultaneously, inhibited,
When rTNF-~ and 1,25-(OH)2D 3 were
however, HL-60 cell growth was synergistically
with the viable cell number beingmaximally
decreased by 39%.
The synergistic effect of rTNF-~ and 1,25-(OH)2D 3 on HL-60 cell differentiation were also observed as shown in Fig. 2.
The HL-60 cell
differentiation was measured by NBT reduction capacity Which has been shown to be the most sensitive index of differentiation (9). After 4 days of treatment with I0 nM 1,25-(OH)2D 3 alone, whereas administration
only 32% of the cells became NBT-positive,
of rTNF-~ alone failed to increase NBT reduction even
at concentrations of up to 1,000 U/ml. and 1,25-(OH)2D3,
however,
The simultaneous addition of rTNF-~
synergistically increased NBT reduction,
with the
NBT-positive cell number maximally increasing by 74%. In the next set of experiments, the expression of c-~yc was examined.
the effect of rTNF-~ and 1,25-(OH)2D 3 on It was found that c-myc mRNk was
preferentially reduced by treatment of the cells with either rTNF-a or 1,25(OH)2D 3 alone.
However,
a significantly greater c-~yc reduction was observed
after combined treatment with rTNF-~ and 1,25-(OH)2D 3 as shown in Fig. 3. Table I shows the result of quantitative measurement of c-myc mRNA.
I00 U/ml
rTNF-~ by itself reduced c-mgc mRNA levels by 55%, and maximal reduction was achieved at this concentration (data not shown). 1153
Inhibition by I0 nM 1,25-
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
L~
14
~
x
10
~ 60
8 .C) c
(0
100
6
T
~-
I
m
4
40
z
[
I
I
I
0
10
100
1000
20 ~0 rl . . . . . . . 0
Q
Q
~. . . . . . . l0
~-- . . . . . 100
--~ 1000
rTNF-c~(U/m£)
Fig. I. S y n e r g i s t i c i n h i b i t i o n of HL-60 c~ll g r o w t h by rTNF-~ and 1,25-(OH)2D 3. HL-60 cells (I x 10 ~ c e l l s / m l ) w e r e c u l t u r e d in the p r e s e n c e of the i n d i c a t e d c o n c e n t r a t i o n s of rTNF-e w i t h (e e) or w i t h o u t (o---o) 10 nM 1,25-(OH)2D 3. A f t e r a 4-day incubation, cell d e n s i t i e s w e r e determined. Each point r e p r e s e n t s the m e a n ± SD of t r i p l i c a t e cultures. Fig. 2. S y n e r g i s t i c s t i m u l a t i o n of HL-60 cell d i f f e r e n t i a t i o n by rTNF-e and 1,25-(OH)2D 3. HL-60 cells (I × I 0 ~ c e l l s / m l ) w e r e c u l t u r e d in the p r e s e n c e of the indicated c o n c e n t r a t i o n s of rTNF-e w i t h (e :) or w i t h o u t (o---o) 10 nM 1,25-(OH)2D 3. After a 4-day incubation, p e r c e n t a g e of NBT r e d u c t i o n - p o s i t i v e cells w e r e d e t e r m i n e d . Each point r e p r e s e n t s the m e a n ± SD of t r i p l i c a t e cultures.
(OH)2D 3 alone was slightly greater (about 62%).
The combined treatment of i00
U/ml rTNF-~ and i0 nM 1,25-(0H)2D 3, however, was more effective, further reducing c-myc mRNA levels by 93%.
a
b
c
d
c-myc 18S-
Fig. 3. S y n e r g i s t i c r e d u c t i o n of c - a y c m R N A by rTNF-~ and 1,25-(OH)2D 3 in HL-60 cells. Total c e l l u l a r R N A was e x t r a c t e d from HL-60 cells after a 20-hr i n c u b a t i o n u n t r e a t e d (a), rTNF-e (100 u/ml) - t r e a t e d (b), 1,25-(OH)2D 3 (10 nM) - t r e a t e d (c), and 1,25-(OH)2D 3 (10 nM) + rTNF-~ (100 U/ml) -t{~ated (d), and was a n a l y z e d by N o r t h e r n b l o t t i n g using a ~ P - l a b e l e d c - m y c probe. 1154
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I
Q u a n t i t a t i v e a n a l y s i s of t h e s y n e r g i s t i c r e d u c t i o n o f c-m~c mRNA by rTNF-~ and 1,25-(OH)2D 3 i n HL-60 c e i l s Additions
c-myc mRNA (cpm)
None
580
rTNF-e
(100 U/ml)
260
1,25-(OH)2D 3 (10 nM)
220
1,25-(OH)2D 3 (10 nM) + rTNF-d (100 U/ml)
40
The cells were treated for 20 hours with rTNF-e (100 U/ml), or 1,25-(OH)2D 3 (10 nM), or with a combination of the two agents. Other details are described under "Materials and Methods". Each value is the mean of three independent experiments.
DISCUSSION While several studies have explored the synergism between rTNF-~ and rIFN-y to induce growth inhibition and differentiation of certain tumor cell lines (3-5), no similar synergism was reported between rTNF-~ and 1,25(OH)2D 3.
In the present study, we have demonstrated for the first time the
synergistic induction of growth inhibition and differentiation after treatment of HL-60 cells with combination of rTNF-~ and 1,25-(OH)2D 3.
This induction
was dose-dependent for rTNF-~ and the combination of optimal concentrations of rTNF-~ and 1,25-(OH)2D 3 synergistically induced growth inhibition and differentation of the cells. Although the events initiating such a process are not known, an early manifestations of growth inhibition and differentiation is the reduction in the expression of c-mgc protooncogene which is greatly amplified in HL-60 cells (8,11-13).
Kelly ~£ aL
(17) have shown a substantial cell cycle-
specific induction of c-myc mRNA in response to mitogens and growth factors. It would appear that high or inappropriate expression of the c-myc gene prevents cell entry into a differentiation program.
In support of this
assertion the present experiments have shown synergism between rTNF-e and
1155
Vol. 152, No. 3, t 9 8 8
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1,25-(OH)2D3 in inducing not only HL-60 cell growth inhibition and differentiation,
but also a complete shut-off of expression of these cells' c-
myc gene. No adequate explanation has yet been provided for the mechanism of the synergism between rTNF-a and 1,25-(OH)2D 3 on HL-60 c-myc reduction.
The rTNF-
a and 1,25-(OH)2D 3 molecules exert different spectra of biological effects, mediated by different kind of receptors; the former acts via cell surface receptors and the latter via cytosolic/nuclear receptors (6,9,18). The cooperative effects of both agents when added together at optimal concentrations,
the levels that caused saturation of the cells' receptors
(9,18), suggest that they reduce c-myc transcription through different molecular mechanisms. The 2n uZgao synergistic antitumor action of rTNF-a and 1,25-(OH)2D 3 observed in the present study suggests that combined treatment with these two natural growth inhibitors might induce better clinical responses and may provide a new approach to the treatment of cancer.
ACKNOWLEDGEMENTS - - W e thank Drs. T. Sugiyama and S. Maeda (Kobe University School of Medicine, Kobe, Japan) for kindly supplying the mouse cmyc cDNA probe.
REFERENCES
I. 2. 3. 4. 5. 6. 7. 8. 9. I0.
Carswell, E.A., Old, L.J., Kassel, R.L., Green, S., Fiore, N. and Williamson, B. (1975) Proc. Natl. Acad. Sci. USA 72, 3666-3670. Williamson, B.D., Carswell, E.A., Rubin, B.Y., Prendergast, J.S. and Old, L.J. (1983) Proc. Natl. Acad. Sci. USA 80, 5397-5401. Sugarman, B.J., Aggarwal, B,B., Hass, P.E., Figari, I.S., Palladino, M.A. and Shepard, H.M. (1985) Science 230, 943-945. Yarden, A. and Kimchi, A. (1986) Science 234, 1419-1421. Trinchieri, G., Kobayashi, M., Rosen, M., Loudon, R., Murphy, M. and Perussia, B. (1986) J. Exp. Med. 164, 1206-1225. Colston, K., Colston, M.J. and Feldman, D. (1981) Endocrinology 108, 1083-1086. Miyaura, C., Abe, E., Kuribayashi, T., Tanaka, H., Konno, K., Nishii, Y. and Suda, T. (1981)Biochem. Biophys. Res. Commun. 102, 937-943. Reitsma, P.H., Rothberg, P.G., Astrin, S.M., Trial, J., Bar-Shavit, Z., Hall, A., Teitelbaum, S.L. and Kahn, A.J. (1983) Nature 306, 492-494. Mangelsdorf, D.J., Koeffler, H.P., Donaldson, C.A., Pike, J.W. and Haussler, M.R. (1984) J. Cell. Biol. 98, 391-398. Matsui, T., Nakao, Y., Kobayashi, N., Kishihara, M., Ishizuka, S., Watanabe, S. and Fujita, T. (1984) Int. J. Cancer 33, 193-202. 1156
Vol. 152, No. 3, 1988
Ii. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Matsui, T., Takahashi, R., Mihara, K., Nakagawa, T., Koizumi, T., Nakao, Y., Sugiyama, T. and Fujita, T. (1985) Cancer Res. 45, 4366-4371. Dalla Favera, R., Wong-Staal, F. and Gallo, R.C. (1982) Nature 299, 6163. Westin, E.H., Wong-Staal, F., Gelmann, E.P., Dalla Favera, R., Papas, T.S., Lautenberger, J.A., Eva, A., Reddy, E.P., Tronick, S.R, Aaronson, S.A. and Gallo, R.C. (1982) Proc. Natl. Acad. Sci. USA 79, 2490-2494. Shirai, T., Yamaguchi, H., Ito, H., Todd, C.W. and Wallace, R.B. (1985) Nature 313, 803-806. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Tronick, S., Aaronson, S. and Leder, P. (1982) Proc. Natl. Acad. Sci. USA 79, 78377841. Kelly, K., Cochran, B.H., Stiles, C.D. and Leder, P. (1983) Cell 35, 603610. Munker, R. and Koeffler, H.P. (1987) Klin. Wochenschr. 65, 345-352.
1157
Vo1.152, No. 3,1988 May 16,1988
COOPERATIVE EFFECTS OF TUMOR NECROSIS FACTOR-~ AND 1,25-DIHYDROXYVITAMIN D 3 ON GROWTH INHIBITION,
DIFFERENTIATION, AND c-myc REDUCTION IN HUMAN
PROMYELOCYTIC LEUKEMIA CELL LINE HL-60*
Yuko Katakami §, Yoshimobu Nakao, Nobuyuki Kat~ami, Tamio Koizumi, Ryoichi Ogawa, Hiroki Yamada, Yoshimi Takai' and Takuo Fujita
Departments of M~dicin~ (Third D i v i s i o n ) and B i o c h e m i s t r y ~ K o ~ U n i v e r s i t y School of Medicine, Kob~ 650, japan Received March 14, 1988 SUMMARY: The simultaneous addition of recombinant human tumor necrosis factor(rTNF-~) and 1,25-dihydroxyvitamin D 3 (1,25-(OH)2D3) synergistically induced growth inhibition and differentiation of the human promyelocytic leukemia cell line HL-60. A significantly greater reduction in c-myc mRNA level was also observed after a 20-h combined treatment with rTNF-~ and 1,25-(OH)2D 3 than was observed following administration of either agent alone. These results suggest that rTNF-~ and 1,25-(OH)2D 3 induce HL-60 cell growth inhibition through different molecular mechanisms, and that their simultaneous administration could provide a new approach to the treatment of cancer. © z988Acaaemic Press,
Inc.
The monokine, TNF-~
, was originally identified by its cytostatic or
cytotoxic effect on some tumor cells (i-3). Due to its apparent tumor selectivity (1-3), intense interest concerning potential clinical application of TNF-~ has been stimulated.
However,
its cytostatic/cytotoxic activity
not all human tumors are sensitive to
(2,3).
Recent studies have shown that the combined treatment of rTNF-a with rlFN-T has a synergistic antiproliferative effect on certain tumor cell lines that are resistant or less sensitive to rTNF-~ (3,4). Synergism between rTNF-
* This investigation was supported in part by a Grant-in Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. § To whom correspondence should be addressed. Abbreviations used are: TNF-~, tumor necrosis factor-a; rTNF-~, recombinant human TNF-e; rIFN-y, recombinant human interferon-y; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; NBT, nitroblue tetrazolium; PBS, phosphate-buffered saline. 0006-291X/88 $1.50 1151
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and rlFN-y to induce differentiation of myeloid leukemia cell lines has also been reported (5).
It is therefore of interest to test for synergism between
rTNF-~ and other naturally occurring growth inhibitors.
One of such growth
inhibitors that has been shown to exert an antiproliferative effect on certain tumor cells is 1,25-(OH)2D 3 (6-11). The human promyelocytic leukemia cell line HL-60 responds to 1,25-(OH)2D 3 by ceasing division and acquiring some of the characteristics of mature myeloid cells, and these phenotypic changes are preceded by the reduction in the expression of the amplified protooncogene, c-myc (8, 11-13).
In the
present studies we have therefore examined the possible cooperative effects of rTNF-~ and 1,25-(OH)2D 3 on growth inhibition,
differentiation,
and reduction
of the level of c-~yc mRNA expression in HL-60 cells.
MATERIALS AND METHODS
~rmf Calf C a ~ - - T h e human promyelocytic leukemia cell line HL60 was kindly provided by Dr. R.C. Gallo (National Cancer Institute, Bethesda, MD). The cells were cultured at 37°C in RPMI-1640 medium (M.A. Bioproduct) supplemented with 25 m~ HEPES buffer, kanamycin (120 Bg/ml), and 10% heatinactivated fetal calf s e r u ~ i n a humidified atmosphere of 5% CO 2 in air. The cells were seeded at 1 x l O J cells per ml in culture flasks and incubated in the presence of various concentrations of rTNF-~ and 1,25-(OH)2D $. The latter was dissolved in ethanol at such a concentration that, when a p p h e d to test cultures, the final concentration of the solvent was less than 0.1%. A control culture medium containing 0.1% ethanol had no effect on HL-60 cell growth or differentiation. /leagea~ - - P u r i f i e d rTNF-~ derived from E s ~ s h g a cof2 (14) was a generous gift from A~ahi Chemical Industry Co., Ltd. It had a specific activity of 2.3 x i0 u units per mg of protein. 1,25-(OH)2D 3 was kindly provided by the Teijin Institute for Biomedical Research. NBT was purchased from Sigma. C a ~ G ~ o w ~ S i MaasaJuz~ar~!--Plastic-adherent cells were detached by gentle pipetting and cell number was determined with a hemocytometer. Viability was determined by the ability of cells to exclude trypan blue. Dif~gaan~££o~ ~ s a g - - D i f f e r e n t i a t i o n was assayed by the ability of cells to reduce NBT to formazan as described previously (ii). Me~sara~aa~ of c-~yc m/97VA--Cells were harvested and washed at 4°C with PBS. Total cellular RNA was extracted by the procedure of Chirgwin at ~ . (15), utilizinR CsCI gradient centrifugation. For Northern blot hybridization, total cellular RNA was denatured, electrophoresed (i0 Bg RNA/lane) in a 1% agarose slab gel, and was transferred to a nitrocellulose filter as described previously (Ii).~^ After baking, the filters were hybridized for 48 h at 42°C with a JzP-labeled DNA probe. The unbound fraction was then removed by extensive washing (ii). The filters were autoradiographed using intensifying screens at -80°C. For dot blot analysis, total cellular RNA samples were dotted onto nitrocellulose filters under a sl@~ vacuum. After baking, the filters were hybridized for 48 h at 42°C with a J~P-labeled DNA probe, and the unbound fraction was then removed by
1152
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
extensive washing. The filters were autoradiographed using intensifying screens for 24 h at -80°C, and the dots were excised and counted in a scintillation counter. Within the range of 1 to I0 ~g the amount of probe hybridized was proportional to the amount of RNA on the filter. The ~ v e l s c-myc mRNA are expressed as cpm per 4 ~g of total cellular RNA. The Plabeled DNA probe used was a nick-translated 5.5-kb Bam HI fragment, containing mouse c-myc gene segments that had been isolated from a mouse plasmacytoma SI07 bacteriophage library and subcloned into pBR322 (16).
of
RESULTS When HL-60 cells were cultured with rTNF-a alone for 4 days, cell growth was slightly inhibited in a dose-dependent manner, with the viable cell number being maximally decreased only to 85% of the control level at concentrations of more than I00 U/ml as shown in Fig. i.
1,25-(OH)2D 3 alone at a
concentration of I0 nM also slightly inhibited HL-60 cell growth and the viable cell number decreased only to 86%. added simultaneously, inhibited,
When rTNF-~ and 1,25-(OH)2D 3 were
however, HL-60 cell growth was synergistically
with the viable cell number beingmaximally
decreased by 39%.
The synergistic effect of rTNF-~ and 1,25-(OH)2D 3 on HL-60 cell differentiation were also observed as shown in Fig. 2.
The HL-60 cell
differentiation was measured by NBT reduction capacity Which has been shown to be the most sensitive index of differentiation (9). After 4 days of treatment with I0 nM 1,25-(OH)2D 3 alone, whereas administration
only 32% of the cells became NBT-positive,
of rTNF-~ alone failed to increase NBT reduction even
at concentrations of up to 1,000 U/ml. and 1,25-(OH)2D3,
however,
The simultaneous addition of rTNF-~
synergistically increased NBT reduction,
with the
NBT-positive cell number maximally increasing by 74%. In the next set of experiments, the expression of c-~yc was examined.
the effect of rTNF-~ and 1,25-(OH)2D 3 on It was found that c-myc mRNk was
preferentially reduced by treatment of the cells with either rTNF-a or 1,25(OH)2D 3 alone.
However,
a significantly greater c-~yc reduction was observed
after combined treatment with rTNF-~ and 1,25-(OH)2D 3 as shown in Fig. 3. Table I shows the result of quantitative measurement of c-myc mRNA.
I00 U/ml
rTNF-~ by itself reduced c-mgc mRNA levels by 55%, and maximal reduction was achieved at this concentration (data not shown). 1153
Inhibition by I0 nM 1,25-
Vol. 152, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
L~
14
~
x
10
~ 60
8 .C) c
(0
100
6
T
~-
I
m
4
40
z
[
I
I
I
0
10
100
1000
20 ~0 rl . . . . . . . 0
Q
Q
~. . . . . . . l0
~-- . . . . . 100
--~ 1000
rTNF-c~(U/m£)
Fig. I. S y n e r g i s t i c i n h i b i t i o n of HL-60 c~ll g r o w t h by rTNF-~ and 1,25-(OH)2D 3. HL-60 cells (I x 10 ~ c e l l s / m l ) w e r e c u l t u r e d in the p r e s e n c e of the i n d i c a t e d c o n c e n t r a t i o n s of rTNF-e w i t h (e e) or w i t h o u t (o---o) 10 nM 1,25-(OH)2D 3. A f t e r a 4-day incubation, cell d e n s i t i e s w e r e determined. Each point r e p r e s e n t s the m e a n ± SD of t r i p l i c a t e cultures. Fig. 2. S y n e r g i s t i c s t i m u l a t i o n of HL-60 cell d i f f e r e n t i a t i o n by rTNF-e and 1,25-(OH)2D 3. HL-60 cells (I × I 0 ~ c e l l s / m l ) w e r e c u l t u r e d in the p r e s e n c e of the indicated c o n c e n t r a t i o n s of rTNF-e w i t h (e :) or w i t h o u t (o---o) 10 nM 1,25-(OH)2D 3. After a 4-day incubation, p e r c e n t a g e of NBT r e d u c t i o n - p o s i t i v e cells w e r e d e t e r m i n e d . Each point r e p r e s e n t s the m e a n ± SD of t r i p l i c a t e cultures.
(OH)2D 3 alone was slightly greater (about 62%).
The combined treatment of i00
U/ml rTNF-~ and i0 nM 1,25-(0H)2D 3, however, was more effective, further reducing c-myc mRNA levels by 93%.
a
b
c
d
c-myc 18S-
Fig. 3. S y n e r g i s t i c r e d u c t i o n of c - a y c m R N A by rTNF-~ and 1,25-(OH)2D 3 in HL-60 cells. Total c e l l u l a r R N A was e x t r a c t e d from HL-60 cells after a 20-hr i n c u b a t i o n u n t r e a t e d (a), rTNF-e (100 u/ml) - t r e a t e d (b), 1,25-(OH)2D 3 (10 nM) - t r e a t e d (c), and 1,25-(OH)2D 3 (10 nM) + rTNF-~ (100 U/ml) -t{~ated (d), and was a n a l y z e d by N o r t h e r n b l o t t i n g using a ~ P - l a b e l e d c - m y c probe. 1154
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Table I
Q u a n t i t a t i v e a n a l y s i s of t h e s y n e r g i s t i c r e d u c t i o n o f c-m~c mRNA by rTNF-~ and 1,25-(OH)2D 3 i n HL-60 c e i l s Additions
c-myc mRNA (cpm)
None
580
rTNF-e
(100 U/ml)
260
1,25-(OH)2D 3 (10 nM)
220
1,25-(OH)2D 3 (10 nM) + rTNF-d (100 U/ml)
40
The cells were treated for 20 hours with rTNF-e (100 U/ml), or 1,25-(OH)2D 3 (10 nM), or with a combination of the two agents. Other details are described under "Materials and Methods". Each value is the mean of three independent experiments.
DISCUSSION While several studies have explored the synergism between rTNF-~ and rIFN-y to induce growth inhibition and differentiation of certain tumor cell lines (3-5), no similar synergism was reported between rTNF-~ and 1,25(OH)2D 3.
In the present study, we have demonstrated for the first time the
synergistic induction of growth inhibition and differentiation after treatment of HL-60 cells with combination of rTNF-~ and 1,25-(OH)2D 3.
This induction
was dose-dependent for rTNF-~ and the combination of optimal concentrations of rTNF-~ and 1,25-(OH)2D 3 synergistically induced growth inhibition and differentation of the cells. Although the events initiating such a process are not known, an early manifestations of growth inhibition and differentiation is the reduction in the expression of c-mgc protooncogene which is greatly amplified in HL-60 cells (8,11-13).
Kelly ~£ aL
(17) have shown a substantial cell cycle-
specific induction of c-myc mRNA in response to mitogens and growth factors. It would appear that high or inappropriate expression of the c-myc gene prevents cell entry into a differentiation program.
In support of this
assertion the present experiments have shown synergism between rTNF-e and
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1,25-(OH)2D3 in inducing not only HL-60 cell growth inhibition and differentiation,
but also a complete shut-off of expression of these cells' c-
myc gene. No adequate explanation has yet been provided for the mechanism of the synergism between rTNF-a and 1,25-(OH)2D 3 on HL-60 c-myc reduction.
The rTNF-
a and 1,25-(OH)2D 3 molecules exert different spectra of biological effects, mediated by different kind of receptors; the former acts via cell surface receptors and the latter via cytosolic/nuclear receptors (6,9,18). The cooperative effects of both agents when added together at optimal concentrations,
the levels that caused saturation of the cells' receptors
(9,18), suggest that they reduce c-myc transcription through different molecular mechanisms. The 2n uZgao synergistic antitumor action of rTNF-a and 1,25-(OH)2D 3 observed in the present study suggests that combined treatment with these two natural growth inhibitors might induce better clinical responses and may provide a new approach to the treatment of cancer.
ACKNOWLEDGEMENTS - - W e thank Drs. T. Sugiyama and S. Maeda (Kobe University School of Medicine, Kobe, Japan) for kindly supplying the mouse cmyc cDNA probe.
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