[Chinese Journal of Cancer 28:2, 117-121; February 2009]; ©2009 Sun Yat-sen University Cancer Center
Basic Research Paper
Comparative proteomics analysis on differentiation of human
promyelocytic leukemia HL-60 cells into granulocyte
and monocyte lineages
Wei-Jia Wang,1 Wei Tang2 and Zong-Yin Qiu1,2,*
1Key Laboratory of Chongqing; Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education; 2Department of Pharmaceutic Analytical Chemistry; Chongqing
Medical University; Chongqing P.R. China
Key words: leukemia, HL-60 cell, differentiation, proteomics
Background and Objective: Human promyelocytic leukemia
cell line HL-60 has potential to differentiate into granulocytes
and monocytes by different inducers, such as NSC67657 and alltrans retinoic acid (ATRA). However, the mechanism is not clear
yet. This study was to induce differentiation of HL-60 cells using
ATRA and NSC67657, and compare the protein expression
patterns using two-dimensional electrophoresis (2-DE). Methods:
HL-60 cells were cultured with ATRA and NSC67657 respectively. Cell proliferation was analyzed by MTT assay. Cellular
surface specific antigen CD11b/CD14 was detected using flow
cytometry (FCM) to assess cell differentiation. The alterations
of cell morphology were observed with cellular chemical staining
under electron microscope. Total protein was separated by modified 2-DE. The differentially expressed proteins were identified
using PD-Quest software and analyzed by MOLDI-TOF MS.
ICAT protein with differential expression was verified by reverse
transcription-polymerase chain reaction (RT-PCR) and western
blot. Results: The granulocytic and monocytic differentiation of
HL-60 cells was induced by ATRA and NSC67657, respectively.
The positive rates of both CD11b and CD14 in HL-60 cells were
over 90% after 5-day treatment (2 μmol/L ATRA or 10 μmol/L
NSC67657); cell morphology also represented characteristics of
differentiation. Proteomic analysis showed that 25 proteins were
differentially expressed with the same pattern in both differentiation groups, ten were differentially expressed only in monocytic
*Correspondence to: Zong-Yin Qiu; Key Laboratory of Chongqing and Key
Laboratory of Laboratory Medical Diagnostics of Ministry of Education; and
Department of Pharmaceutic Analytical Chemistry; Chongqing Medical University;
Chongqing 400016 P.R. China; Tel.: 86.23.68485312; Fax: 86.23.68485005;
Email: zongyin141@sina.com
Submitted: 05/12/08; Revised: 08/25/08; Accepted: 10/23/08
This paper was translated into English from its original publication in Chinese.
Translated by: Guangzhou Liheng and Wei Liu on 12/15/08.
The original Chinese version of this paper is published in: Ai Zheng (Chinese
Journal of Cancer), 28(2); http://www.cjcsysu.cn/cn/article.asp?id=14948
Previously published online as a Chinese Journal of Cancer E-publication:
http://www.landesbioscience.com/journals/cjc/article/8424
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differentiation group and 15 only in granulocytic differentiation
group. Among them, ICAT expression was upregulated during
NSC67657-induced differentiation of HL-60 cells. Conclusion:
A batch of differentially expressed proteins has be found by 2-DE
in HL-60 cells with granulocytic and monocytic differentiation,
which would contribute to the following functional research on
related proteins.
Inducing differentiation of tumor cells has become a major
strategy in tumor therapy. Great achievements have been made
in searching for clinically practical and effective drugs to induce
differentiation and apoptosis of tumor cells.1 Since 1990s, alltrans retinoic acid (ATRA) has become another hot spot in
medical research after arsenic trioxide (As2O3). Up to now,
ATRA is a classic and widely used drug in treating leukemia.
Used alone or in combination with As2O3, ATRA shows obvious
effect on leukemia. Recently, National Cancer Institute (NCI)
of the United States has developed a new steroid inducer
NSC67657, with a chemical name of (3R, 8R, 9S, 10R, 13S)10,13-dimethyl-17-[(R)-5-(methylsulfonyl-oxy)pentan-2-yl]
hexadecahydro-1H-cyclopenta[α]phenanthren-3-yl methane
sulfonate. Different from ATRA, NSC67657 induces only monocytic differentiation of acute promyelocytic leukemia HL-60
cells.2 Our study was to identify differentially expressed proteins
during monocytic and granulocytic differentiation, as well as specific
proteins and potential drug targets in each differentiating direction.
Materials and Methods
Cell line and reagents. Human acute promyelocytic leukemia
cell line HL-60 was purchased from Shanghai Cell Bank.
NSC67657 was kindly offered by National Cancer Institute of the
United States. ATRA was purchased from Sigma Co. RPIM-1640
medium was purchased from Gibco Co. Fetal bovine serum (FBS)
was purchased from Hyclone Co. (USA). DNase I and RNase were
purchased from TaKaRa Co. (Dalian); protease inhibitor Cocktail,
NBT staining kit, anti-ICAT polyclonal antibody and anti-βtubulin monoclonal antibody were purchased from Santa Cruz Co.
Anti-CD11b, anti-CD14 antibody and corresponding control
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Comparative proteomics analysis on differentiation of human promyelocytic leukemia HL-60 cells into granulocyte and monocyte lineages
Figure 1. The proliferation of HL-60 cells treated by NSC67657 and ATRA
of different concentrations.
antibodies were purchased from Jingmei Biotech Co. (Shenzhen).
pH3-10 non-linear IPG gel strip, urea, 2-directional electrophoresis reagents (such as CHAPS) and silver staining reagent kit were
purchased from Bio-Rad Co.
MTT assay for the detection of HL-60 cell proliferation.
HL-60 cells at logarithmic growth phase were suspended at the
density of 1 × 105/mL with culture medium containing 10%
FBS, and treated with ATRA (1, 2 and 3 μmol/L) and NSC67657
(5, 10, 15 and 20 μmol/L), respectively. Untreated cells were set
as control. Culture plates (with three wells for each group) were
incubated at 37°C in 5% CO2 for 24, 48, 72 and 96 h, respectively. Cells were then cultured with MTT reagent (20 μL/well) for
4 h, centrifuged at 3800 ×g for 10 min, and treated with dimethyl
sulfoxide (DMSO, 150 μL/well) for 5 min. The absorbance at
490 nm (A490) was measured with spectrophotometer. Proliferation
inhibition rate was calculated with the formula: 1 - (A490 of treatment well/A490 of control well) × 100%.
Flow cytometry for measurement of CD11b/CD14 expression on HL-60 cells. HL-60 cells treated with 2 μmol/L ATRA
or 10 μmol/L NSC67657 for five days, and untreated HL-60 cells
at logarithmic growth phase were collected, washed with PBS for
three times, and suspended at the density of 1 × 106/mL. FITClabeled anti-CD14 antibody and control antibody were then added
for the detection of CD14 expression by flow cytometry.
Microscopic observation of HL-60 cell differentiation.
HL-60 cells were treated as stated in subsection 1.3, washed with
PBS for three times, suspended at the density of 5 × 105/mL,
centrifuged to prepare smears and underwent Wright’s staining,
NBT and esterase staining and NaF inhibition test as described
in related literature.3 Cell morphology was observed under
118
Figure 2. Morphology of HL-60 cells before and after treatment of
NSC67657 and ATRA. (A) After treatment of NSC67657 and ATRA,
griscent–black precipitation can be found in HL-60 cells (NAE × 400). (B)
The griscent–black precipitation is weak in untreated HL-60 cells (NAE ×
400). (C) After 5-day treatment of NSC67657, HL-60 cells present monocytic differentiation with irregular nuclei and hypochromatic endochylema
(arrow) (Wright’s × 400). (D) After 5-day treatment of ATRA, HL-60 cells
present granulocytic differentiation (arrows) (Wright’s × 400). (E) After
treatment of NSC67657 and ATRA, cyan-black precipitation can be seen
in the endochylema of positive HL-60 cells (NBT × 400). (F) The cyan-black
precipitation is weak in untreated HL-60 cells (NBT × 400).
microscope; positive cells in three successive visual fields were
counted to calculate positive rate.
Two-dimensional electrophoresis (2-DE) for protein separation in HL-60 cells. HL-60 cells at logarithmic growth phase
were collected in 2-mL tubes (2 × 107/tube), and underwent three
freezing-thawing cycles in liquid nitrogen, then added with cytolysis [modified formulation containing 8.5 mol/L urea, 2 mol/L
thiourea, 4% CHAPS, 65 mmol/L DTT, 0.2% (w/v) Bio-Lyte
ampholine (pH3-10), 40 mmol/L Tris, 20 μg/mL Mg2+-activated
DNase, 5 μg/mL RNase and protease inhibitor Cocktail] (1 mL/
tube) and placed on ice for 30 min. At this time, no intact cells could
be seen under microscope. The cell lysate was then centrifuged at
15 000 ×g and 4°C for 60 min to collect supernatant for quantitative analysis with standard curves. The supernatant (200 μg, 330
μL) was loaded onto the IPG gel strip, underwent iso-electric
focusing (IEF) and SDS-PAGE electrophoresis. IEF conditions
were as follows: 450 V for 2 h, 4380 V for 2 h and 4380 V
for 93 min. To enhance sensitivity of the detection, separated
protein spots were visualized with silver staining, and analyze by
PDQuest software. Those spots with a density difference of more
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2009; Vol. 28 Issue 2
Comparative proteomics analysis on differentiation of human promyelocytic leukemia HL-60 cells into granulocyte and monocyte lineages
Figure 3. Differential expression of proteins in HL-60 cells before and after treatment of NSC67657 and ATRA. The differentially expressed protein spots
have been marked by cycles.
than 2-fold as compared with control were considered differentially expressed protein spots.
Matrix-assisted laser desorption/ionization-time of flight mass
spectrometry (MALDI-TOF MS) for analysis of differentially
expressed protein spots. Differentially expressed protein spots on
silver-stained gel strip were precisely cut off to prepare samples for
mass spectrometry according to the modified method described
by Hellman et al.4 Saturated CHCA solution was prepared with
CH3CN and 5% TFA (1:1, v/v). Peptide mixture was dissolved
in 2 μL CH3CN and 5% TFA (1:1, v/v), then mixed with 2 μL
saturated CHCA solution. Sample solution/matrix solution (1 μL)
was added onto plates. These plates were dried at room temperature for 8 h.
Mass spectrometry was performed by Voyager DE Pro Matrix
Assisted Laser Desorption/Ionization-Time of Flight Mass
Spectrometer (ABI, US) at the following setting: reflector mode;
positive ion detection; N2 laser wavelength of 377 nm; ion accelerating voltage of 20 kV; pulse length of 3 ns; ion delay time of 100 ns;
grid voltage of 75%, conducting wire of 0.003%; laser intensity
of 850–1200 internal units; spectrometric signal intensity was the
accumulation of 150 scans.
Protein sequences were identified by searching Swissprot and
NCBInr database using MASCOT searching engine. Match was
deemed significant when the score was above 55.
Verification of differential expression of β-catenin interacting
protein ICAT at transcription and translation levels. HL-60 cells at
logarithmic growth phase were collected and suspended at the density
of 1 × 106/mL, treated with or without 10 μmol/L NSC67657 at
37°C and 5% CO2 for five days. Total RNA was extracted, and
underwent reverse transcription and 30 cycles of amplification. The
products underwent electrophoresis on 0.2 g/L agarose gel. Optical
density was measured with Bio-Rad gel imaging system.
HL-60 cells of NSC67657 group and control group were
collected to prepare cell lysate using cytolysis. Total protein
was extracted and quantitatively measured with Bradford
method; 60 μg of the protein underwent electrophoresis on
16% SDS-PAGE, then transferred, blocked and ultured with antibody. Chemiluminescence assay was performed and optical density
was measured with Bio-Rad gel imaging system. The above experiments were repeated three times.
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Statistical analysis. The data were analyzed with one-way
analysis of variance using SPSS11.5 software. Differences were
considered significant when p < 0.05.
Results
HL-60 cell proliferation after different treatments. At varied
concentrations, both NSC67657 and ATRA inhibited proliferation
of HL-60 cells, with stronger inhibition at higher concentration
(Fig. 1). Since most cells were dead at extremely high concentration, 10 μmol/L NSC67657 and 2 μmol/L ATRA were selected for
the establishment of cell models.
CD11b/CD14 expression and morphology of HL-60 cells
after treatment. When treated with either NSC67657 or ATRA,
the percentage of differentiated HL-60 cells increased with time.
After 5-day treatment, the positive rate of CD14 was (96.3 ±
3.68)% in NSC67657 group, that of CD11b was (94.9 ± 3.17)%
in ATRA group.
With Wright’s staining, granulocytic cells and monocytic cells
remarkably increased in both NSC67657 and ATRA groups,
while HL-60 cells of control group were still immature and
under-differentiated; NBT-stained cells could be seen in both
NSC67657 and ATRA groups, more in ATRA group while less in
NSC67657 group (p < 0.05, Fig. 2). With esterase staining, the
positive rates were (69.35 ± 6.43)% before NaF inhibition and
57.5% after NaF inhibition in ATRA group, with an inhibition
rate of < 50%; the positive rates were (81.75 ± 9.22)% before NaF
inhibition and 11.7% after NaF inhibition in NSC67657 group,
with an inhibition rate of > 50%, suggesting that differentiation
was successfully induced.
Differentially expressed proteins in treated and untreated
HL-60 cells. A total of 1102 protein spots were found in control
cells, 1064 in NSC67657 group and 988 in ATRA group.
Performing MALDI-TOF MS, 50 differentially expressed proteins
were identified. Among them, 25 showed the same variation
tendency in both differentiation directions; then only differentially
expressed in monocytic differentiation; 15 only differentially
expressed in granulocytic differentiation (Table 1).
Verification of differentially expressed protein ICAT. As
compared with that in control group, the expression of Icat gene
was significantly upregulated in NSC67657 group (p < 0.05),
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Comparative proteomics analysis on differentiation of human promyelocytic leukemia HL-60 cells into granulocyte and monocyte lineages
Table 1
The differentially expressed proteins in
HL-60 cells before and after inducement
Figure 4. Verification of differential expression of ICAT gene and protein
in HL-60 cells by RT-PCR (A) and western blot (B). Lane 1, untreated HL-60
cells; lane 2, NSC67657-treated HL-60 cells; lane 3, ATRA-treated HL-60
cells. ap < 0.05, bp < 0.01, vs. control.
while it remained unchanged in ATRA group; the expression of
ICAT protein showed the same variation (Fig. 4).
Discussion
HL-60 is an ideal cell line for research on hematologic diseases,
with a major advantage of the potential of multi-directional differentiation under the action of inducers. Moreover, it has reliable
cellular chemical markers that reflect its functions, especially the
enhancement of NBT reduction that reflects phagocytic ability
and the up-regulation of CD11b and CD14 expression, which are
well-recognized markers for granulocytic and monocytic differentiation.5 With the potential of bi-directional differentiation of
HL-60 cells and the establishment of cellular model of differentiation, we used proteomic technique 2-DE to identify critical
proteins that are closely related to differentiation of these cells as
well as potential targets for new drugs, and identified 50 differentially expressed proteins. Differential expression of ICAT protein
was verified at both transcription and translation levels for the
sake of identifying differentiation-related molecules among these
differentially expressed proteins.
In this study, we used low-voltage non-linear separation to
separate total protein in HL-60 cells. The modified method has
three prominent advantages over the traditional method. Firstly,
it produces excellent separation, and no interventions that damage
the proteins or cause protein loss are needed. Secondly, it saves
time. During traditional IEF electrophoresis, the voltage increasing
and focusing processes need close monitoring. The desalting,
voltage increasing and focusing processes take at least 16–22 h,
which causes much inconvenience in time management. Moreover,
constant high voltage of 500 V will lead to some unfavourable
consequences, such as protein degradation. At a lower voltage,
the modified method achieves desalting and focusing within
8 h in HL-60 cells or ordinary blood cells. It takes only two days
with the modified method to finish the separation, while the
traditional method takes four days. Last but not the least, with
lower voltage, less heat will be produced. At the current of 10 μA,
low-voltage method generates only 50% of the heat as the tradi120
Chinese Journal of Cancer
2009; Vol. 28 Issue 2
Comparative proteomics analysis on differentiation of human promyelocytic leukemia HL-60 cells into granulocyte and monocyte lineages
tional method does. Therefore, heat-caused protein degradation,
which is one major reason for the stripe and shadow on gel, will
be minimized.
With 2-DE and MADLI-TOF analysis, 50 differentially
expressed protein spots were identified. β-catenin-interacting
protein ICAT, an inhibitor in Wnt/β-catenin pathway, inhibits
transcription of down-stream genes by hindering the binding of
β-catenin to T-cell factor (TCF), and plays important roles in
the development and progression of tumor.6,7 Other proteins
identified in our study included oncogene-encoded proteins, such
as TPMS,8 proteins encoded by tumor suppressor gene, such
as CUTL1, TRIT1 and CLDN23,9-11 proteins related to the
differentiation of tumor cells, such as ICAT, FYN and USF2,12-14
apoptosis-related proteins, such as NME2 and BCL2L1515,16 and
proteins related to cellular skeleton or protein phosphorylation,
which might be indispensable for the differentiation of cells. Of
course, the differential expression of these proteins needs to be
verified with western blot assay.
Acknowledgements
Grant: Foundation for Great Scientific Research in Chongqing
(No. 2004-27).
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