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Endocrinology Vol. 138, No. 2 805-809
Copyright © 1997 by The Endocrine Society

Articles

3,5,3'-Triiodo-L-Thyronine Potentiates All-Trans-Retinoic Acid-Induced Apoptosis during Differentiation of the Promyeloleukemic Cell HL-60

Satoru Suzuki, Hiroaki Kobayashi, Rieko Sekine, Mieko Kumagai, Michiaki Mikoshiba, Jun-ichiro Mori, Masahiro Hara, Kazuo Ichikawa and Kiyoshi Hashizume

Department of Geriatrics, Endocrinology, and Metabolism, Shinshu University School of Medicine, Asahi, Matsumoto, Japan

Address all correspondence and requests for reprints to: Satoru Suzuki, M.D., Department of Geriatrics, Endocrinology, and Metabolism, Shinshu University School of Medicine, 3–1-1 Asahi, Matsumoto, Nagano 390, Japan. E-mail: rounen3{at}gipac.shinshu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the programmed cell death mediated by thyroid hormone is not well evaluated in mammalian cells, thyroid hormone plays a crucial role in differentiation of the cells during the metamorphosis of Xenopus, suggesting that thyroid hormone has the potential ability to induce the apoptosis. To investigate the thyroid hormone-inducible apoptosis, we cultured HL-60 cells with various amounts of all-trans-retinoic acid (RA) and L-T3. T3 alone did not induce the apoptosis of the cells. T3, however, suppressed the proliferation of cells in the presence of RA. DNA ladder and microscopical examination showed that the reduction of cell number was due to the apoptosis induced by RA. These findings suggested that T3 affects the apoptotic process during the differentiation of HL-60 cells by RA. T3-inducible apoptosis may require the factors augmented by RA in HL-60 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS plays a major role in the development of the central nervous system, the immune system, and carcinogenesis (1). Apoptosis occurs through the activation of a cell-intrinsic suicide program. The basic machinery to carry out apoptosis appears to be present in essentially all mammalian cells at all times, but activation of the suicide program is regulated by many different signals that originate from both intracellular and extracellular factors (2). During apoptosis, the nucleus and cytoplasm condense, and the dying cell often fragments into membrane-bound apoptotic bodies that are rapidly phagocytosed and digested by macrophages or neighboring cells (3, 4).

In the tadpole, thyroid hormone (T3) plays a crucial role in morphological development during growth (5). During development, programmed cell death and differentiation of the cells take place in the presence of T3 (6). This observation suggests that T3 is important not only in differentiation, but also in apoptosis.

The actions of T3 and retinoic acid (RA) are initiated through the interaction of the receptors with the specific DNA sequences in the regulatory regions (7). It is understandable that inducible genes by T3 and/or RA may control the growth and differentiation of the cells. Treatment of the pituitary cell line, GH4C1, with RA and T3 potentiates gene expression of GH in these cells, suggesting synergic regulatory functions of these ligands (8).

In the present study, we demonstrated that the T3-dependent apoptosis is present in HL-60 cells, and that the apoptosis requires RA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
HL-60 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan) and were cultured in RPMI supplemented with 10% FBS. We distributed the cells to adjust 1 x 104 cells/ml into 10-cm petri dishes when we started the experiments in this study.

After the indicated times, 0.2 ml of cells was taken to make cytospin preparations, which were fixed in 100% methanol and stained with May-Giemsa solution (Kanto Chemical Co., Tokyo, Japan).

Cell counts
The number of cells was counted after staining with trypan blue. Cells were scored as differentiated if they showed features of metamyelocytes or more differentiated forms and as apoptotic if there was evidence of nuclear pycnosis and fragmentation, cytoplasmic condensation, and basophilia, as previously described (9). Morphological assessment of apoptosis was performed in a blinded fashion, without revealing the initial treatment assignment. Differential counts (at least 200 cells/sample) were performed on the cytospin preparations. All results in Tables 1Go and 2Go were expressed as the mean ± SD of three independent experiments, each performed in duplicate.


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  		Table 1. Distribution of cells

 

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  		Table 2. Percentage of NBT-positive cells

 
T3 binding assay
The binding of [125I]T3 to isolated nuclei was determined using nuclei prepared from HL-60 cells cultured in 10-cm petri dishes. Approximately 3 x 107 cells were harvested and homogenized in 2 ml SMTD buffer (0.32 M sucrose, 1 mM MgCl2, 2 mM dithiothreitol, and 20 mM Tris, pH 7.5). The homogenate was centrifuged at 1000 x g for 10 min, and resultant nuclear pellet was incubated briefly with 1 ml SMTD buffer in the presence of 0.25% Triton X-100 (vol/vol). After washing twice with 5 ml SMTD buffer, the nuclear pellet was resuspended in SMTD buffer and incubated with [125I]T3 at 20 C for 2 h. After incubation, the samples were centrifuged at 1000 x g for 10 min. The nuclear pellet was washed twice with 1 ml SMTD buffer. The nuclear-bound [125I]T3 was determined by counting the radioactivity of the nuclear pellet with a {gamma}-spectrometer. The dissociation constant and the maximal binding capacity of [125I]T3 nuclear binding were estimated by the method of Scatchard (10).

Nitroblue tetrazolium (NBT) reduction assays
The cells were grown in the medium with the indicated concentration of agents for 5 days, and 2 x 105 cells were aliquoted into 0.2 ml RPMI medium with 0.8 ml 0.125% NBT and 20 µM of 12-O-tetradecanoylphorbol-13-acetate. The cells were incubated at 37 C for 30 min, centrifuged at 1100 x g for 7 min at room temperature, and resuspended in 200 µl PBS. Cytospins from aliquots of the samples were stained with Safranin-O (for 5 min). The percentage of NBT-positive cells in each preparation was determined.

DNA fragmentation
After growing for 3 days in the presence or absence of the indicated hormones, 2 x 106 cells were lysed in 100 µl DNA isolation buffer (10 mM Tris-HCl, pH 8.0; 10 mM EDTA; and 0.5% Triton X-100). After 10-min incubation at 4 C, cell lysates were spun down. The supernatant was transferred and incubated for 1 h at 37 C with 400 µg/ml ribonuclease A. Twenty microliters of 5 M NaCl and 120 µl isopropanol were added after incubation for 1 h at 37 C with 400 µg/ml proteinase K. Precipitated fragments were dissolved in 20 µl TE buffer. Five microliters of the aliquot were taken for measurement of the concentration of DNA to adjust the contents of DNA for application to 1.6% agarose gel.

Measurement of DNA contents in cells
The amount of DNA was determined by the method of Burton (11), with calf thymus DNA as the standard.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thyroid hormone receptors are present in HL-60 cells
T3 binding to nuclear fraction was determined by incubating whole nuclei of HL-60 cells with various concentrations of [125I]T3 for 2 h at 20 C. The approximate number of binding sites was estimated to be 1000 sites/cell, with a Ka of 3 x 109 M (Fig. 1Go). The maximal binding capacity was not altered by the treatment of cells with RA during incubation for 24 h.



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  		Figure 1. Effects of RA on the nuclear binding of [125I]T3 in HL-60 cells. Scatchard plots of the nuclear binding of [125I]T3 in the cells after 24-h incubation. {blacksquare}, 0.1% ethanol; {square}, 10-6 M RA.

 
Apoptosis is induced by T3 in HL-60 cells 5 days after incubation with RA, but not without RA
The number of cells was counted 3 and 5 days after the beginning of incubation with 1 x 10-6 M RA and/or T3. As shown in Fig. 2Go, there was no significant difference between the number of cells treated with and without T3 in the absence of RA. Treatment with RA alone suppressed the proliferation of the cells 5 days after incubation. When cells were incubated with RA and T3, the suppression was stronger than that obtained in the cells treated with RA alone. As shown in Fig. 3Go, exponentially growing cultures had frequent mitotic figures and morphological features characteristic of a blastic leukemic cell line. T3 alone did not affect them morphologically. RA-induced differentiation of HL-60 cells is associated with the appearance of apoptotic cells. Apoptotic HL-60 cells can be recognized by the appearance of nuclear condensation and fragmentation, increased basophilia of the cytoplasm, and a marked decrease in cellular volume. Combined treatment with RA and T3 was significantly more active than RA alone in inducing apoptosis; cultures treated with 10-6 M T3 and 10-6 M RA showed extensive accumulation of apoptotic cells (Table 1Go). NBT reduction assays demonstrated that 10-6 M T3 did not affect the number of positive cells in the presence of 10-6 M RA, as shown in Table 2Go.



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  		Figure 2. Growth curves for HL-60 cells treated with T3 and/or RA at 10-6 M. The indicated agents were added on day 0, and cell number was determined on days 3 and 5. The values shown are the mean ± SD for three separate experiments.

 


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  		Figure 3. Effects of RA and/or T3 on the morphology of HL-60 cells. a, Untreated HL-60 cells. b, HL-60 cells treated with 10-6 M RA; arrows indicate typical differentiated cells with segmented nuclei. c, HL-60 cells treated with 10-6 M T3. d, HL-60 cells treated with 10-6 M T3 and RA; arrows show typical apoptotic cells with fragmented nuclei. Magnification, x500.

 
T3 induces DNA fragmentation in HL-60 cells 3 days after incubation with RA
DNA fragmentation is one of the specific findings in the apoptosis. As shown in Fig. 4Go, DNA fragmentation occurred 3 days after incubation of HL-60 cells with RA and T3. No significant changes were observed in electrophoretic patterns between the cells treated with ethanol as a control and those treated with 10-6 and 10-5 M T3 alone.



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  		Figure 4. DNA fragmentation in T3- and/or RA-treated HL-60 cells. Five micrograms of cellular DNA were extracted and subjected to electrophoresis on a 1.6% agarose gel to detect nucleosome laddering.

 
T3 decreases cell number in a dose-dependent manner in the presence of RA
We incubated the cells with different concentrations of T3 and RA for 5 days. In the medium containing 10% FCS, T3 alone did not show significant effects on the numbers of the cells, as shown in Fig. 5Go. T3 increased, rather than decreased, the numbers of the cells in T3-stripped medium. RA alone diminished the numbers of the cells, and the maximal effect of RA was obtained at 10-6 M RA or higher.



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  		Figure 5. T3 with RA suppress the number of the cells in a dose-dependent manner. The effects of various concentration of T3 and RA were evaluated in these experiments. After 5 days of incubation with the indicated concentrations of compounds, the number of cells was counted. All data were corrected for the number of cells treated with ethanol as the control. • and {blacksquare}, The numbers of the cells treated with T3 alone, in normal medium and T3-stripped medium, respectively. {blacktriangleup}, {dagger}, {square}, and {circ}, The numbers of the cells treated with various concentrations of T3 in the presence of 10-10, 10-8, 10-6, and 10-5 M RA, respectively. The values shown are the mean ± SD for three separate experiments. T3 concentrations are given on the abscissa, and the relative number of cells is shown on the ordinate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis is known to associate with the differentiation of several organs, including the immune system and the central nervous system (12). Carcinogenesis is also associated with apoptosis (13). Numerous agents that induce apoptosis have been reported. Glucocorticoid is one of the factors that induces the apoptosis of lymphocytes. Some genes that are regulated by the activation of glucocorticoid receptor (GR), one of the nuclear receptors, may contribute to the induction of apoptosis in GR-presenting cells. However, Helmberg et al. (14) demonstrated that the apoptosis was also induced by glucocorticoid in GR-mutated cells. Thus, the mechanisms of glucocorticoid-induced apoptosis are controversial and are dependent not only on GR, but also on other factors.

The action of thyroid hormone is initiated through the binding of thyroid hormone receptors (TR) to the specific sequences in the regulatory gene, which resembles the mechanism of the action of glucocorticoid (15, 16). Apoptosis induced by T3, however, has not been well investigated. In the tadpole, the epidermis consists of three different cell types: apical, basal, and skein. The skein cells of the tadpole finally disappear to form the apical border of the adult skin (17). T3 selectively suppresses the mitosis of skein cells, whereas the proliferation of basal cells is initiated by T3, implying that T3 induces the apoptosis of skein cells during metamorphosis (5). T3 induces apoptotic cell death during transforming growth factor-{alpha}-induced hemopoiesis (18). These findings suggest that T3 induces apoptosis in some cells, by functional modification of extra- and intracellular signaling other than T3.

As Scatchard analysis demonstrated that the TRs were present in nuclei of HL-60 cells, as reported previously (19), T3 potentiation of RA action is possibly mediated by TR activation. However, more studies are needed to verify this point.

T3 alone did not affect the NBT reduction of HL-60 cells, whereas RA did. Further, T3 did not increase the number of NBT-positive cells in the presence of 10-6 M RA. Ballerini et al. (20) observed T3-induced differentiation of HL-60 cells in the presence of RA. They, however, showed that the effect of T3 was not clear in the presence of 10-7 M RA or higher, as 10-7 M RA induced maximal activation in differentiation. We used 10-6 M RA in the NBT assay. Thus, the effect of T3 on differentiation was not obvious in this study.

We demonstrated no significant effect of T3 alone on the number of HL-60 cell in culture, whereas RA decreased the number. Combined treatment of T3 and RA further reduced the number of the cells, indicating that T3 potentially augments the RA-induced decrease in cell number.

Microscopic examinations showed fragmented nuclei in the presence of RA, but T3 alone did not show any fragmentation of nuclei. Further, the DNA fragmentation assays showed a ladder pattern of the DNA in the presence of RA. However, the ladder was not observed after incubation with T3 alone. These results indicate that the decrease in the cell number is due to the apoptosis, but not to the necrosis.

Apoptosis and differentiation by retinoids and vitamin D3 are well known. It is reported that induction of apoptosis requires ligand activation of endogenous RXRs and that 9-cis-RA and vitamin D3 together promote apoptosis of HL-60 cells (9, 21). All-trans-RA not only activates RA receptors, but also binds to and activates RXRs (9). Furthermore, 9-cis-RA may be produced by isomerization of the ligand. Thus, the apoptotic events caused by the combined treatment with RA and T3 may be mediated by RXRs in these experiments.

As mentioned above, T3 alone did not affect the number of cells. On the other hand, the RA-induced decrease in cell number was augmented by T3. The influence of T3 was observed even when the concentration of RA was reduced to 1 x 10-10 M.

Clinically, the congenital defect in the thyroid gland causes severe brain damage, short stature, and immature faces, namely cretinism (22). T3 deprivation in the adult, however, does not cause these abnormalities. Thus, although T3 action is manifested through the activation of the same receptors, the phenotypic response by T3 may be different in mature and immature cells. HL-60 cells, which are considered immature, are differentiated by RA treatment, implying that induction of apoptosis by T3 is initiated during the development or differentiation of HL-60 cells.

The mechanisms are unknown. Hypothetically, T3 potentially has the ability to induce apoptosis of HL-60 cells, and RA may augment the expression of T3 receptors to increase T3 responsiveness. RA up-regulates the expression of RARs in HL-60 cells, and T3 also increases the expression of TRß1 isoforms in GH3 cells (9, 23). However, Scatchard analysis demonstrated that the number of T3 receptors was not altered by RA at least within 24 h of incubation. Thus, there are two possibilities to interpret in the induction of apoptosis by T3. One is that the factors contributing to cell death may be controlled by RA and T3 synergistically, as several reports demonstrate that T3 and RA potentiate synergic regulatory functions (8, 24). The second is that the induction of apoptosis by T3 may require the cofactors that are regulated by RA. The precise mechanisms of T3-induced acceleration of RA-induced apoptosis, however, remain to be elucidated.

Received September 16, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Adrends MJ, Wyllie AH 1991 Apoptosis: mechanisms and role in pathology. Int Rev Exp Pathol 32:223–254[Medline]
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  5. Kanamori A, Brown DD 1993 Cultured cells as a model for amphibian metamorphosis. Proc Natl Acad Sci USA 90:6013–6017[Abstract/Free Full Text]
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  9. Nagy L, Thomazy VA, Shipley GL, Fesus L, Lamph W, Heyman RA, Chandraratna RAS, Davies PJA 1995 Activation of retinoid X receptors induces apoptosis in HL 60 cell lines. Mol Cell Biol 15:3540–3551[Abstract]
  10. Scatchard G 1949 The attraction of protein for small molecules and ions. Ann NY Acad Sci 51:660–672[CrossRef]
  11. Burton K 1956 Study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–323[Medline]
  12. Thompson CB 1995 Apoptosis in the pathogenesis and treatment of disease. Science 267:1456–1461[Abstract/Free Full Text]
  13. Graeber TG, Osmanian C, Jack T, Housman DE, Koch CJ, Lowe SW, Giaccia AJ 1996 Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors. Nature 379:88–91[CrossRef][Medline]
  14. Helmberg A, Auphan N, Caelles C, Karin M 1995 Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor. EMBO J 14:452–460[Medline]
  15. Larsen PR, Harney JW, Moore DD 1986 Sequences required for cell-specific thyroid hormone regulation of rat growth hormone promoter activity. J Biol Chem 261:14733–14736[Abstract/Free Full Text]
  16. Carson-Jurica MA, Schrader WT, O’Mally BW 1990 Steroid receptor family: structure and function. Endocr Rev 11:201–220[Abstract/Free Full Text]
  17. Robinson DH, Heintzelman MB 1987 Morphology of ventral epidermis of Rana catesbeiana during metamorphosis. Anat Rec 217:305–317[CrossRef][Medline]
  18. Gandrillon O, Ferrand N, Michaille JJ, Roze L, Zile MH, Samarut J 1994 c-erbA{alpha}/T3R and RARs control commitment of hematopoietic self-renewing progenitor cells to apoptosis or differentiation and are antagonized by the v-erbA oncogene. Oncogene 9:749–758[Medline]
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