Jin W et al / Acta Pharmacol Sin 2004 Mar; 25 (3): 319-326
Wei JIN2, Le-feng QU3, Ping MIN2, Shan CHEN2, Hong LI4, He LU2,4, Yong-tai HOU2, 5
2Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203;
3Department of Vascular Surgery & Institute of Vascular Surgery of PLA, Changhai Hospital, Shanghai 200433;
4Le pôle franco-chinois du science du vivant et génomique, Ruijin Hospital, Shanghai 200025, China
1 Project supported by the National Natural Science Foundation of China, (30200151), and One Hundred People Programs of Chinese Academy of Sciences (2001).
5 Correspondence to Dr Yong-tai HOU. Phn 86-21-5080-6600, ext 2416. Fax 86-21-5080-6029. E-mail ythou@mail.shcnc.ac.cn
Received 2003-06-06 Accepted 2003-11-11
KEY WORDS HL-60 cells; homoharringtonine; apoptosis; oligonucleotide array sequence analysis; gene expression
ABSTRACT
AIM: To identify genes responsive to apoptosis in HL-60 cells treated by homoharringtonine. METHODS: cDNA microarray technology was used to detect gene expression and the result of microarrays for genes (TIEG and VDUP1) was confirmed by Northern analysis. RESULTS: Seventy-five individual mRNAs whose mass changed significantly were identified. Among these genes (25 were up-regulated and 50 were down-regulated), most are known related to oncogenes and tumor suppressor. Some genes were involved in apoptosis signaling pathways. CONCLUSION: TGFβ and TNF apoptosis signaling pathways were initiated during apoptosis in HL-60 cells. TIEG and VDUP1 play important roles in mediating apoptosis.
INTRODUCTION
Homoharringtonine (HHT) is a cytotoxic alkaloid isolated from the evergreen tree Cephalotaxus harringtonia native to the southern provinces of China. It can stop cell cycle by blocking cell G1 into S phase and from G2 into M phase. Clinical studies have indicated that HHT is effective in treating acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), but not acute lymphoblastic leukemia (ALL) nor solid tumors[1]. HHT has been used alone and in combination with interferon-alpha or low-dose cytarabine in late and early chronic phases of CML patients, with positive results[2].
HHT induced apoptosis in HL-60 cells when the cells were exposed to 1×10-7 mol/L HHT for 4 h. DNA extracted from treated cells showed a typical internuc-leosomal DNA degradation. This effect of HHT was shown to appear in a concentration- and time-dependent manner. These results suggest that antitumor mechanism of HHT is related to its apoptosis inducing activity[3]. In HL-60 cells, HHT predominantly inhibited protein synthesis compared to RNA and DNA synthesis[4].
In an attempt to identify genes responsive to HHT-induced apoptosis and to reveal apoptosis signaling pathways, cDNA microarray technology was used to simultaneously display changes of gene expression. In all, ten microarrays were screened and each array representing 14218 human genes. Among the 75 genes characterized, most are previously known as cancer-related genes. A few are novel genes that may be involved in apoptosis signaling pathways.
MATERIALS AND METHODS
Cell cultures Human leukemia HL-60 cells were cultured in RPMI-1640 medium containing 10 % bovine serum in 95 % air and 5 % CO2 at 37 ºC. For the experiment, HL-60 cells were incubated with 4.64 mg/L HHT (Beijing Union Pharmaceutical Factory) or RPMI 1640 medium alone for 15 min, 1 h, 3 h, 6 h, 24 h.
Detection of apoptosis DNA fragmentation The total cellular DNA was extracted from HL-60 cells untreated and treated with HHT for 15 min,1 h, 3 h, 6 h, 24 h by the method described by Slin and Stafford with some slight modifications[5] . In brief, cells were washed in phosphate-buffered saline (PBS) and lysed overnight at 37 ºC in lysis buffer containing Tris-HCl 10 mmol/L (pH 8.0), edetic acid 10 mmol/L, 0.4 % sodium dodecyl-sulfate, and proteinase K 100 mg/L. After complete digestion, saturated phenol was added to the cell lysates and mixed fully. Samples were then centrifuged for 5 min. Chloroform was added to the supernatant isolated from the previous step, mixed fully and centrifuged as above. Supernatant was mixed with 2.5-fold volume of absolute ethanol and NaCl at 0.2 mol/L final concentration for DNA precipitation. The DNA pellets were obtained by centrifugation for 10 min and then air-dried, dissolved in TE buffer containing RNase 0.5 g/L (Sigma Chemical Co) at 37 ºC for 30 min. Electrophoresis was performed on 1.5 % agarose gel. The DNA was visualized by UV illumination.
RNA isolation Total RNA from HL-60 cells was extracted according to the original Chomczynski method with slight modifications[6]. Cells were collected and homogenized in Solution D containing 1 % β-mercapto-ethanol. After centrifugation, supernatant was extracted with phenol:chloroform (1:1) twice and acidic phenol:chloroform (5:1) once. The RNA from aqueous phase was precipitated by cold isopropanol and dissolved in deionized H2O (Milli-Q). Messenger RNAs were purified using an Oligotex-dT mRNA Midi Kit (Qiagen, Inc, Carlsbad, CA).
Construction of microarray and probe preparation The microarray was
constructed according to Brown's method (in: http://cmgm.stanford.edu/pbrown/protocols/index.html).
The 14218 microarray consists of 14218 full-length or partial complementary
DNAs (cDNAs) representing novel, known and control genes provided by United
Gene Holdings, Ltd (1111 Zhongshan Bei Er Road, Shanghai, China). The known
genes were selected from National Center for Biotechnology Information (NCBI)
Unigene set and cloned into PBS plasmid vector. The control spots of non-human
origin included rice U2 RNA gene (8 spots), Hepatitis C Virus
(HCV) coat protein gene (8 spots) and spotting solution alone (32 spots). The
cDNA inserts were amplified by PCR using universal primers to the plasmid vector
sequences. All PCR products were examined by agarose gel electrophoresis to
ensure the quality and the identity of the amplified clones as expected. The
PCR products were dissolved in a buffer containing 3×0.15 mol/L NaCl and
0.015 mol/L sodium citrate (SSC) solution. The solution were spotted onto silylated
slides (CEL Associates, Houston TX) using a Cartesian PixSys 7500 motion control
robot (Cartesian Technologies, Irvine, CA) fitted with ChipMaker Micro-Spotting
Technology (TeleChem International, Sunnyvale, CA). Glass slides with spotted
cDNA were then hydrated for 2 h in 70 % humidity, dried for 0.5 h at room temperature,
UV cross-linked (65 mj /cm). They were further processed at room temperature
by soaking in 0.2 % sodium dodecyl sulfate (SDS) for 10 min, distilled H2O
for 10 min, and 0.2 % sodium borohydride (NaBH4) for 10 min. The
slides were dried and ready for hybridization.
The fluorescent cDNA probes were prepared through reverse transcription of the isolated mRNAs and then purified according to Schena et al[7,8]. The mRNA samples from the control cells were labeled with Cy3-dUTP (Amersham Pharmacia Biotech) and those from treated cells with Cy5-dUTP (Amersham Pharmacia Biotech). The two color probes were then mixed, precipitated with ethanol and dissolved in 20 µL of Hybridization Solution (5×SSC, 0.4 % SDS, 50 % formamide and 5×Denhardt's Solution).
Hybridization and washing of microarray Microarrays were pre-hybridized with Hybridization Solution containing 0.5 g/L denatured salmon sperm DNA at 42 ºC for 6 h. Fluorescent probe mixtures denatured at 95 ºC for 5 min were applied onto the pre-hybridized microarrays under cover glasses. After the microarrays were hybridized at 42 ºC for 15-17 h, they were washed at 60 ºC for 10 min each in solutions of 2×SSC and 0.2 % SDS; 0.1×SSC and 0.2 % SDS; 0.1×SSC, and then dried at room temperature.
Detection and analysis of microarray The microarrays were scanned with a ScanArray 3000 (GSI Lumonics, Bellerica, MA) at two wavelengths to detect emission from both Cy3 and Cy5. The acquired images were analyzed using ImaGene 3.0 software (BioDiscovery, Inc, Los Angeles, CA). The intensities of each spot at the two wavelengths represent the quantity of Cy3-dUTP and Cy5-dUTP, respectively, hybridized to each spot. Ratio of Cy5 to Cy3 was computed for each location on each microarray. Overall intensities were normalized with a correction coefficient obtained using the ratio of 40 housekeeping genes (list of these genes is available at http://www.biodoor.com/). Genes were identified as differentially expressed if the ratio of Cy5/Cy3 was >2 or <0.5. To minimize artifacts arising from low expression values, only genes with raw intensity values for both Cy3 and Cy5 of >800 counts were chosen for differential analysis.
Northern analysis RNA (Northern) blots were carried out with mRNA extracted from the control and treated cells. The probes were labeled with 32P (Beijing Furi bio-technology Ltd) using the random primer method. The blots were scanned with a cyclone instrument (Molecular Dynamics, Sunnyvale, CA), and the data were analyzed with Parkard 3.0 software (Packard Instruments, Meriden, CT).
RESULTS
HHT induced apoptosis in HL-60 cells Apoptosis induced by HHT in HL-60 cells was examined (Fig 1). DNA agarose gel electrophoresis showed that HL-60 cells presented the typical DNA ladder pattern of apoptosis after treatment with HHT 4.64 mg/L for 6 h and 24 h.
Fig 1. Patterns of DNA fragmentation during HHT induced apoptosis in HL-60 cells. 1) Lambda DNA/Eco130I (StyI) marker (bp); 2) untreated cells; 3) HHT 4.64 mg/L, 15 min; 4) HHT 4.64 mg/L, 1 h; 5) HHT 4.64 mg/L, 3 h; 6) HHT 4.64 mg/L, 6 h; 7) HHT 4.64 mg/L, 24 h.
cDNA microarray analysis In order to identify both early and late apoptosis-responsive genes, mRNA was isolated from cells treated with HHT for 15 min, 1 h, 3 h, 6 h, and 24 h and subjected to microarray hybridization. RNA samples from cells treated with RPMI 1640 medium were used as the control. A total of ten microarrays were screened. Two microarrays were screened for each parallel mRNA sample at each time point. The hybridization results from all ten microarrays were compiled and sorted on the basis of fold change compared to control cells. The average ratio of the two microarrays screened at each time point was adopted. Genes that displayed approximately two-fold or greater changes were scored as significant changes. Seventy-five mRNA species out of the 14218 genes were identified by these criteria. Of these 75 mRNA species, 25 were up-regulated and 50 were down-regulated at different time points (Tab 1).
Tab 1. List of differentially expressed genes responsive to HHT-induced apoptosis.
|
Symbol |
GenBank |
15 min |
1 h |
3 h |
6 h |
24 h |
Gene
name |
|
|
|
ratio |
ratio |
ratio |
ratio |
ratio |
|
| TIEG* |
NM_005655 |
1.54 |
4.41 |
12.45 |
29.21 |
1.00 |
Transcription
factor; TGFB inducible early growth response |
| ACVRL1* |
NM_000020 |
2.91 |
7.83 |
8.549 |
0.55 |
0.47 |
Activin A receptor type II-like 1 |
| PPP1CA* |
NM_002708 |
1.53 |
2.28 |
4.27 |
5.64 |
1.48 |
Protein phosphatase 1, catalytic subunit, alpha isoform |
| IMMT* |
NM_006839 |
0.95 |
0.48 |
2.21 |
5.82 |
0.88 |
Inner membrane
protein, mitochondrial (mitofilin) |
| BRD2* |
NM_005104 |
1.39 |
2.63 |
4.68 |
6.48 |
1.68 |
Bromodomain containing 2 |
| NFKBIA* |
NM_020529 |
1.70 |
1.93 |
5.73 |
8.77 |
1.45 |
Nuclear factor
of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha |
| DUSP6* |
NM_001946 |
0.73 |
2.26 |
2.23 |
3.92 |
0.47 |
Dual specificity
phosphatase 6 |
| NFIL3* |
NM_005384 |
0.64 |
1.37 |
2.01 |
3.44 |
0.56 |
Nuclear factor,
interleukin 3 regulated |
| RAB14* |
NM_016322 |
0.60 |
0.38 |
1.26 |
3.24 |
0.89 |
RAB14, member
RAS oncogene family |
| VAV1* |
NM_005428 |
0.98 |
0.47 |
1.51 |
5.07 |
1.15 |
Vav 1 oncogene |
| SLC |
AB018010 |
1.48 |
1.57 |
4.82 |
7.65 |
1 |
Solute carrier family 3, member 2 |
| TNFAIP3* |
NM_006290 |
1.56 |
1.78 |
2.34 |
5.61 |
1.33 |
Tumor necrosis factor, alpha-induced protein 3 |
| RBM4* |
NM_002896 |
1.12 |
1.16 |
2.91 |
5.54 |
1.03 |
RNA binding motif protein 4 |
| TAGLN2 |
D21261 |
1.15 |
0.44 |
1.29 |
3.85 |
0.99 |
Transgelin
2 |
| FAP48 |
U73704 |
1.36 |
2.31 |
2.64 |
3.67 |
0.94 |
FKBP-associated protein |
| PPIE |
AF042385 |
0.88 |
0.49 |
1.39 |
3.6 |
0.73 |
Peptidylprolyl isomerase E (cyclophilin
E) |
| RRM1* |
X59543 |
0.97 |
0.38 |
1.02 |
3.42 |
0.71 |
Ribonucleotide
reductase M1 polypeptide |
| ABH |
NM_006020 |
1.09 |
2.07 |
2.68 |
3.25 |
1.08 |
Alkylation
repair; alkB homolog |
| HLA-A |
D32129 |
0.61 |
0.29 |
0.79 |
2.89 |
0.35 |
Major histocompatibility complex,
class I, A |
| M9 |
NM_004690 |
1.46 |
2.35 |
2.26 |
2.81 |
1.57 |
Muscle specific gene |
| ATP |
X69908 |
0.62 |
0.27 |
0.96 |
2.58 |
0.44 |
ATP synthase, H+ transporting,
mitochondrial F0 complex, subunit c, isoform
2 |
| TKT |
L12711 |
0.5 |
0.34 |
0.81 |
2.02 |
0.33 |
Transketolase
(Wernicke-Korsakoff syndrome) |
| VDUP1* |
S73591 |
0.65 |
1.86 |
6.03 |
11.81 |
0.35 |
Thioredoxin
interacting protein |
| C-MYC* |
K02276 |
0.82 |
2.85 |
0.62 |
0.3 |
|
C-MYC, oncogene |
| IDH3B |
AK001905 |
0.87 |
0.38 |
0.76 |
1.89 |
0.43 |
Isocitrate
dehydrogenase 3 (NAD+) beta |
| LAMR1 |
U43901 |
0.57 |
0.41 |
1.02 |
1.8 |
0.31 |
Laminin receptor 1 (ribosomal protein
SA) |
| RPS9 |
AL080243 |
0.52 |
0.33 |
0.79 |
1.78 |
0.35 |
Ribosomal protein S9 |
| FDPS |
D14697 |
0.58 |
0.43 |
0.83 |
1.74 |
0.46 |
Dimethylallyltranstransferase
(geranyltranstransferase) |
| DLEC1 |
AL137706 |
0.4 |
0.22 |
0.65 |
1.44 |
0.44 |
Deleted in lung and esophageal cancer 1 |
| GNB |
M24194 |
0.49 |
0.69 |
0.73 |
1.41 |
0.28 |
Guanine nucleotide binding protein (G protein), beta polypeptide
2-like 1 |
| SAT |
AL050290 |
0.33 |
0.69 |
0.93 |
1.4 |
0.26 |
Spermidine/spermine
N1-acetyltransferase |
| SLC |
J03592 |
0.45 |
0.44 |
0.69 |
1.15 |
0.29 |
Solute carrier family 25, member 6 |
| MVD |
NM_002461 |
0.47 |
0.5 |
0.72 |
1.15 |
0.3 |
Mevalonate
(diphospho) decarboxylase |
| RPL8* |
Z28407 |
0.46 |
0.47 |
0.58 |
0.98 |
0.42 |
Ribosomal protein L8 |
| BC-2 |
AF042384 |
0.38 |
0.49 |
0.52 |
0.96 |
0.33 |
Putative breast adenocarcinoma
marker |
| HIP2 |
U58522 |
0.43 |
0.43 |
0.56 |
0.91 |
0.38 |
Huntingtin
interacting protein 2 |
| PSMB5 |
D29011 |
0.44 |
0.47 |
0.49 |
0.85 |
0.34 |
Proteasome
(prosome, macropain) subunit, beta
type, 5 |
| YWHAE |
U54778 |
0.41 |
0.58 |
0.49 |
0.82 |
0.33 |
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation
protein |
| TAF7 |
U18062 |
0.49 |
1.07 |
0.71 |
0.78 |
0.4 |
TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated
factor |
| SNRPA1 |
NM_003090 |
0.4 |
0.93 |
0.67 |
0.77 |
0.31 |
Small nuclear ribonucleoprotein
polypeptide A’ |
| RPS |
M84711 |
0.46 |
0.74 |
0.51 |
0.76 |
0.32 |
Ribosomal protein S |
| SLC |
J02683 |
0.46 |
1.08 |
0.74 |
0.74 |
0.29 |
Solute carrier family 25, member 5 |
| ACLY |
X64330 |
0.49 |
0.89 |
0.61 |
0.73 |
0.32 |
ATP citrate lyase |
| SGCB |
U31116 |
0.37 |
0.74 |
0.54 |
0.65 |
0.27 |
Sarcoglycan,
beta (43kD dystrophin-associated glycoprotein) |
| ANP32B |
NM_006401 |
0.46 |
0.73 |
0.45 |
0.64 |
0.27 |
Acidic (leucine-rich) nuclear phosphoprotein 32 family, member B |
| FEN1 |
AC004770 |
0.65 |
0.47 |
0.45 |
0.64 |
0.34 |
Flap structure-specific endonuclease
1 |
| NCL |
M60858 |
0.53 |
0.74 |
0.4 |
0.63 |
0.24 |
Nucleolin
|
| |
|
|
|
|
|
|
|
| TRA1 |
X15187 |
0.55 |
0.49 |
0.41 |
0.6 |
0.27 |
Tumor rejection antigen (gp96) 1 |
| SSB |
X69804 |
0.56 |
1.07 |
0.55 |
0.59 |
0.41 |
Sjogren syndrome antigen B (autoantigen
La) |
| PSMA2 |
D00760 |
0.72 |
1.2 |
0.46 |
0.59 |
0.29 |
Proteasome
(prosome, macropain) subunit, alpha
type, 2 |
| DAD1* |
NM_001344 |
0.39 |
1.01 |