Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 243-247

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 243-247

Conformational Changes in HL60 Cells during Differentiation and Apoptosis Induced by Genistein*

WANG Zhao ** , ZHOU Jiangbing, SUN Suqin †, LIU Mingjie , ZHANG Hongjun

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
†Department of Chemistry, Tsinghua University, Beijing 100084, China

* Supported  partly by the Tsinghua University - Hong Kong Baptist University Joint Institute for Research of Chinese Medicine and by the “985" Fund from Tsinghua University  
 ** To  whom correspondence should be addressed, E-mail:  zwang@tsinghua.edu.cn  

Received: 2000-10-20

Code Number: ts01074

Abstract:   

Genistein can induce not only differentiation but also apoptosis in HL60 human leukemia cells. During the differentiation and apoptosis, the HL60 cells undergo some conformational changes. Fourier transform infrared (FT-IR) was employed to detect those changes. The results showed that the contents of DNA/protein and glycoprotein/protein increased. The a-helix of the membrane protein also increased. In addition, the C-O (H) stretching mode of serine, threonine and tyrosine residues of the cell proteins also changed. Those conformational changes suggest some mechanisms for how genistein affects the HL60 cells.

Key  words:  genistein; HL60; conformational change; differentiation; apoptosis

Introduction   

Genistein is a naturally occurring phytoestrogen present in a variety of plant foods, including soybean[1]. It is known to inhibit both tyrosine protein kinases and DNA topoisomerase II. Akiyama et al.[2]  described the ability of genistein to specifically inhibit protein tyrosine kinase (PTKs), presumably at the level of the ATP binding site. PTKs seem to play a key role in tumorigenesis and are known to be associated with growth control receptors such as epidermal growth factors[3] , platelet-derived growth factors[4] , insulin[5]  and insulin-like growth factors, as well as with several oncogene products[6]. The genistein to modulation of PTKs is, therefore, assumed to affect cell proliferation. Genistein is known to inhibit the growth of many tumor cells, including human leukemia cell line HL60[7,8]. The study of the effect of genistein on the HL60 cell line focused on its ability to induce cell differentiation and apoptosis in the past few years[7,8]. Recently, Fourier transform infrared (FT-IR) spectroscopy with different measurement modes has been widely used to investigate biological systems. FT-IR has become a powerful tool for analysis of cell components, such as membranes[8]  , proteins[9]  and nucleic acids[10] , as well as for complex biological materials, such as tissues[11]  and microbiological systems[12-14]. Other studies have reported on differences between normal and various types of tumor cells, including skin[10]  and leukemia cells[15].

In this study, the functional changes of HL60 cells induced by genistein were studied in vitro. Then, FT-IR was used to study the conformational changes of HL60 treated with genistein.

1 Materials and Methods  

1.1 Materials

HL60 cells were obtained from the Biology Department of Beijing Normal University. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco Laboratories (Grand Island, NY, U.S.A.). Nitrobluetetrazolium (NBT), thiazolyl blue (MTT) (analytical grade) genistein, TPA, and supreptomycin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

2 Cell culture

Cells were grown in  RPMI 1640  medium supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin (100 mg/mL) at  37 °C  in culture chamber containing 5% CO₂ .

1.3 NBT reduction test

The percentage of HL60 cells capable of reducing NBT was determined by counting the number of cells that contained precipitated formazan particles after the cells were incubated with NBT (1.0 mg/mL) at  37 °C  for 30 min. TPA (200 ng/mL) was used as a stimulator for the formation of formazan.

1.4 Phagocytosis test

Cells (1x10⁶) were suspended in serum-free RPMI 1640 medium containing  0.2%  latex particles (average diameter was 0.806 mm; Sigma Chemical) and incubated at  37 °C  for 4 hours. After incubation, the cells were washed once with phosphate-buffered saline (PBS). The cells containing more than 10 latex particles were scored as phagocytic cells.

1.5 Flow cytometry assay

Apoptosis cells were determined using an ApoAlert Annexin V-FITC Apoptosis Kit. All the operations were performed according to the User Manual. Briefly, about 1x10⁵ - 1x10⁶ cells were rinsed with binding buffer and were resuspended in  200 mL  of the binding buffer. Then 5 mL of Annexin V and 10 mL of Propidium Iodide were added to the suspension. The cells were incubated at room temperature for 5-15 min in the dark, and then analyzed by flow cytometry (Coulter, EPICS ELITE, U.S.A.) at 488 nm.

1.6 In situ detection of DNA cleavage by the TUNEL procedure

Apoptotic cells were also determined using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) kit (Sino-America, China). Briefly, cells were fixed in freshly prepared 10% formaldehyde solution for 25 min at room temperature. After rinsing with PBS, the cells were permeated with 0.2% Triton X-100 and then incubated for 1 hour at  37 °C  with TdT and Biotin-II-dUTP to label the cleaved DNA. Then the cells were stained with DAB and hematoxylin solution. Cells showing a positive reaction (containing brown granules in the cell nucleus) were scored.

1.7 Treatment of cells for FT-IR detection 

Cells used for studying the changes were treated by various concentrations of genistein for 5 days. Then cells were washed, resuspended in  0.9%  NaCl solution and deposited on IR-transparent CaF₂-windows that were then kept in a desiccator under a mild vacuum and dried down as thin circular disk films with diameters of 2-3 mm[15].

1.8 FT-IR spectroscopic study

An FT-IR (Perkin Elmer, Spectrum GX, USA) equipped with a 5-beam condenser was used to measure the IR spectra of the treated and control cells previously deposited on IR-transparent CaF2-windows. The FT-IR spectra were collected with a resolution of 4 cm^-1  using 32 scans. All the data was collected with the Windows software offered by the Perkin Elmer Co. [15].

2 Results  

2.1 Differentiation and apoptosis of HL60 cells

NBT test results showed that HL60 cells were induced to differentiate along granulocytic and monocyte/macrophage lineage after being treated with genistein (Fig.1). After treatment with genistein at a concentration of 30 mmol/L for 5 days, 48.3% of the treated cells showed NBT positive, while 47.3% of the HL60 cells treated with 0.5 mmol/L all trans-retinoic acid (ATRA) showed NBT positive and 8.7% of the untreated HL60 cells showed NBT positive. Twenty eight percent of the treated cells could uptake latex beads (Fig.2), indicating that percentage of treated differentiated phagocytic cells. Four point eight percent and 19.7% of the cells in the  untreated and  0.5 mmol/L  ATRA treated media were positive.

Genistein not only induced differentiation in HL60 cells, but also induced apoptosis in HL60 cells. The FACS assay was used to monitor HL60 cells treated with 30 mmol/L genistein for 3 days. The results in Fig.3 show that genistein induced apoptosis in the HL60 cells. Sixteen percent of the cells were stained only by Annexin V, while only 4.4% of the untreated cells were stained only by Annexin V. The TUNEL assay results confirmed the apoptosis. The apoptosis of HL60 cells treated with genistein at this concentration detected by TUNEL assay occurred in 16.5% of the cells.

2.2 Conformational changes of HL60 cells

During the differentiation and apoptosis induced by genistein, HL60 cells underwent great conformational changes which could be detected by FT-IR. Those changes included changes of the structures of the DNA, the membrane proteins, and the glycoproteins.

The band at 1087 cm^-1  refers to the symmetric PO2^-1(v_sPO₂^-) stretching vibration of phosphate groups that are parts of the DNA backbone[16]. Often the A1087/A1540 ratio is used to illustrate the change of the cell DNA/protein content. Increases of this ratio imply increased DNA/protein content[17]. In this experiment, the A1087/A1540 ratio increased with genistein treatment to values, higher than that of untreated HL60 cells (Fig.4), indicating that the cell DNA/protein content increased with genistein treatment.

The membrane protein secondary structure also underwent slight changes with genistein treatment. The amide I band is often employed to study the membrane protein structure[18]. The peaks for a-helix, b-sheet, and random coil are located at 1649-1660 cm^-1 , 1615-1637 cm^-1  and 1638-1648 cm^-1 , respectively[19]. Treatment with 40 mmol/L genistein, the amide I peak of the HL60 cells moved from 1648 cm^-1  to 1651 cm^-1  (data not reported), implying that the a-helix in the membrane had increased.

The glycoprotein content of HL60 cells also changed with genistein treatment. The glycoprotein peak is located in the spectra between 1180 cm^-1  and 980 cm^-1 [20]. Glycoprotein content changes are studied using the glycoprotein/amide II[20] . After treatment with genistein, the glycoprotein/amide II ratio increased (Fig.5) suggesting that the glycoprotein/protein ratio increased in the HL60 cells treated by genistein.

A  distinct change also appeared in the 1164 cm^-1  band which reflects the stretching mode of serine, threonine and tyrosine residues of the cell proteins[21]. In tumor cells, the band is moved to a higher wavenumber, so the band at 1164 cm^-1  is weaker than that in normal cells, because most of the hydrogen bonds connecting different  C-O(H)s are destroyed[22]. In the HL60 cells, this band was absent or less prominent, but after treatment with genistein, this peak became apparent  (Fig.5).

3 Discussion  

Genistein had been reported to induce cellular changes for several years. It can  induce HL60 cells to differentiate along granulocytic and monocyte/macrophage lineage, as judged by biochemical markers. Also, genistein can induce HL60 cell apoptosis as confirmed by flow cytometry and TUNEL assay[7,8]. Genistein treatment caused conformational changes in the HL60 cells. Genistein induced not only differentiation in the HL60 cells but also apoptosis. The DNA of cells that underwent apoptosis was decomposed, but the  DNA/protein ratio of HL60 cells increased. Thus, some proteins in the HL60 cells treated with genistein must have broken down. Also the conformational structure of the HL60 cells membrane proteins could be induced by genistein to change from random coil (1648 cm^-1 ) to a-helix (1651 cm^-1 ) as a result of genistein-protein interaction. The mechanism of this conversion induced by genistein is not well understood. However, the carbonyl group of genistein is supposed to interact with the NH or OH group of certain protein components, which contribute to the helical structure so that the a-helix structure increases while the random coil decreases[23-25]  . In addition to these changes, the  C-O(H)  stretching mode of the serine, threonine and tyrosine residues of the cell proteins increased, probably indicating that some proteins in the cell membrane changed after combining with genistein. All the conformational changes provide information about the mechanism of genistein inhibition with HL60 cells.

References

1. Peterson G, Barnes S. Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene. Biochem Biophys Res Commun, 1991, 179: 661-667.
2. Akiyama T, Ishida J, Nakagawa S, et al. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem, 1987, 262: 5592-5595.
3. Ushiro H, Cohen S. Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cell membranes. J Biol Chem, 1980, 255: 8363-8365.
4. Nishimura J, Huang J S, Duel T F. Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes. Proc Natl Acad Sci, USA, 1982, 79: 4303-4306.
5. Petruzelli LM, Ganguly S, Smith C, et al. Insulin activates a tyrosine-specific protein kinase in extracts of 3T3-L1 adipocytes and human placenta. Proc Natl Acad Sci, USA, 1982, 79: 6792-6797.
6. Rubin J B, Shia M A, Pilch PF. Stimulation of tyrosine-specific phosphorylation in vitro by insulin-like growth factor I. Nature, 1983, 305: 438-440.
7. Constantinou A, Kiguchi K, Huberman E. Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein. Cancer Res, 1990, 50 (9): 2618-2624.
8. Frank Traganos, Barbara Ardelt, Nadine Halio, et al. Effects of genistein on the growth and cell cycle progression of normal human lymphocytes and human leukemia MOL-4 and HL-60 cells. Cancer Res, 1992, 52: 6200-6208.
9. Surewica W K, Mantsch H H. New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochim Biophys Acta, 1988, 952(2): 115-130.
10. Taillandie R E, Liquier F. Infrared spectroscopy of DNA. Methods in Enzymol, 1992, 211: 307-335.
11. Choo L P, Jackson M, Halliday W C, et al. Infrared spectroscopic characterisation of multiple sclerosis plaques in the human central nervous system. Biochim Biophys Acta, 1993, 1182(3): 333-337.
12. Hong K, Sun S, Tian W, Chen G Q, et al. A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy. Appl Microbiol Biotechnol, 1999, 51: 523-526.
13. Cheung H Y, So C W, Sun S. Interfering mechanism of sodium bicarbonate on spore germination of Bacellus stearothermiphilus. J Appl Microbiol, 1998, 84: 619-626.
14. Cheung H Y, Cui J, Sun S. Real-time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier-transform infrared spectroscopy during germination. Microbiol, 1999, 145: 1043-1048.
15. Schultz Christian P, Liu Kan-Zhi, Johnston J B, et al. Study of chronic lymphocytic leukemia cells by FT-IR spectroscopy and cluster analysis. Leukemia Res, 1996, 30(8): 649-655.
16. Taillandie R E, Liquier F. Infrared spectroscopy of DNA. Methods in Enzymol, 1992, 211: 307-335.
17. Benedetti E, Teodori L, Trinca M L, et al. A new approach to the study of human solid tumor cells by means of FT-IR microspectroscopy. Appl Spectrosc, 1990, 44: 1276-1285.
18. Haris P I, Chapman D. Analysis of peptide and protein structures using Fourier transform infrared spectroscopy. Meth in Mol Biol, 1994, 22:  183-202.
19. Neault J F, Tajmir-Riahi H A. Interaction of cisplatin with human serum albumin, drug binding mode and protein secondary structure. Biochim Biophys Acta, 1998, 1384(1): 153-159.
20. Wang Jan-jen, Chi Chin-wen, Lin Shan-yang, et al. Conformation changes in gastric carcinoma cell membrane protein correlated to cell viability after treatment with adamantyl maleimide. Anticancer Res, 1997, 17: 3473-3478.
21. Susi H. In Structure and Stability of Biological Macromolecules Marcel Dekker. New York, 1969, 575-663.
22. Wong P T T, Codrin M, French S W. Distinctive infrared spectral features in liver tumor tissues of mice: evidence of structural modifications at the molecular level. Exp Mol Pathol, 1991, 55:  269-284.
23. Przbycien T M, Beiley J E, Structure-relationships in the inorganic salt-induced precipitation of alpha-chumo trypsin. Biochim Biophys Acta, 1989, 995: 231-245.
24. Kin Y, Rose CA, Liu Y, et al. FT-IR and near-infrared FT-Raman studies of the secondary structure of insulinotropin in the solid state: a-helix to b-sheet conversion induced by phenolo and/or high shear force. J Pharm Sci, 1994, 83: 1175-1180.
25. Wong P T T, Wong R K, Caputo T A, et al. Infrared spectroscopy of exfoliated human cervical cells: evidence of extensive structural changes during carcinogenesis. Proc Natl Acad Sci, USA, 1991, 88: 10.

Copyright 2001 - Tsinghua Science and Technology

The following images related to this document are available:

Photo images