ISSN 1672-9145 Acta Biochimica et Biophysica Sinica 2005, 37(2): 107–112 CN 31-1940/Q Construction and Co-expression of Bicistronic Plasmid
Encoding Human WEE1 and Stem Cell Factor
Ping LEI, Wen-Han LI, Wen-Jun LIAO, Bing YU, Hui-Fen ZHU, Jing-Fang SHAO, and Guan-Xin SHEN*
Laboratory of Molecular and Immuno-Pharmacology, Department of Immunology and Department of Pharmacology, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430030, China
Abstract To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and
promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy.
In the prent study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein超越自己
human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and
enhanced green fluorescent protein (EGFP) parately with internal ribosome entry site (IRES). We first
examined the expression and location of WEE1Hu in Chine hamster ovary (CHO) cells and showed that
WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We
determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells,
which were proved by rever transcription polymera chain reaction (RT-PCR) and flow cytometry,
respectively. Furthermore, inhibition of cisplatin-induced apoptosis was obrved in CD34+ cells transfected
with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our
study suggests that the introduction of two functional genes via bicistronic vector is more powerful and
efficient than single gene therapy.
Key words internal ribosome entry site (IRES); human WEE1 (WEE1Hu); stem cell factor (SCF)
The clinical dos of chemotherapeutic agents or gamma rays are limited by the bone marrow suppression produced in anti-tumor therapy. How to protect hematopoietic stem cells (HSCs) from damage to alleviate marrow toxicity and achieve greater antitumor effects propo an attractive issue.In respon to DNA damage, WEE1Hu can inactivate the Cdc2/CyclinB complexes to activate the G 2 DNA da-
mage checkpoints and inhibit the G 2/M transition in the cell cycle to prevent cells from progressing through to mitosis prior to completion of repair, which subquently results in an irreversible alteration to the genome that can have profound effects on cellular viability and coordinated cell proli
feration and development [1–3]. WEE1 may rve as a key regulator of both HIV type 1 Vpr- and gamma irradiation-mediated apoptosis and possibly rve as a general regulator linking the cell cycle to some pathways of apoptosis [4].
Stem cell factor (SCF), known as the ligand for c-kit,plays a central role in normal HSC development and func-tion during embryogenesis and in adult life. In vitro , stem cell factor promotes the survival of hematopoietic pro-genitors and enhances their proliferation in respon to specific growth factors. SCF is expresd from two al-
insistedternatively spliced transcripts as a transmembrane precursor. Its extracellular N-terminal globular domain interacts with its receptor resulting in the proliferation and differentiation of hematopoietic cells [5,6].
Herein, we demonstrate the construction of the
bicistronic vector and bioactivity estimations of expresd
proteins, which are the ability of SCF to bind to its recep-
tor and the anti-apoptotic function of WEE1.
Materials and Methods
Received: November 18, 2004 Accepted: January 5, 2005
This work was supported by a grant from the National Natural Science
Foundation of China (No. 30170879
Abbreviations: HSC, hematopoietic stem cell; SCF, stem cell factor;
IRES, internal ribosome entry site
*Corresponding author: Tel, 86-27-83692640; Fax, 86-27-83693500; E-mail, myjsz@mails.tjmu.edu
at Chine Academy of Medical Sciences on January 5, 2014
108 Acta Biochim Biophys Sin V ol. 37, No. 2Cloning the SCF gene The SCF extracellular domain cDNA (mhSCF) without the termination codon was amplified by rever transcrip-
tion polymera chain reaction (RT-PCR) from total RNA prepared from HepG2 cells using the n (P1: 5'-CTG-CGTCGAC Sal I CACCA 184TGAAGAAGACACAAACT-3')and the antin (P2: 5'-GACAGGATCC Bam HI C 789TATT-ACTGCTACTGC-3') primers. Kozak translation initiation site, Sal I and Bam HI sites were introduced into primers,respectively.The amplified mhSCF product was digested with the appropriate restriction enzymes, and the resulting frag-ments were subcloned into the pEGFP-N1 vector (BD
Biosciences, CA, USA) to produce pEGFP-N1/mhSCF which is in frame with the EGFP coding quence. Se-quencing was performed using P1 and P2 primers (BioAsia,Shanghai, China).Construction of recombinant bicistronic expression vector pWISG pCMV-WEE1 (kindly provided by Dr. Hiroto Okayama,Osaka University) was digested with Bam HI and cloned into Bam HI cut pET-28a to produce pET-28a-WEE1.Then, pWI was constructed by ligating an Nhe I plus Eco RI cut fragment of pET-28a-WEE1 into the pIRES (BD Biosciences ). Sal I plus Not I were ud to exci the pEGFP-N1/mhSCF. The resulting fragment was ligated into the pWI to produce pWISG. T
he structure of the cloned bicistronic expression castte containing the WEE1gene followed by IRES quence and mhSCF/EGFP gene
is shown in Fig. 1.Cell culture and transfection procedure The Chine hamster ovary cell line CHO was ud in the experiments. Cells were cultured in RPMI 1640 medium (Gibco BRL, NY, USA) supplemented with 10% FBS
(Gibco BRL) at 37 °C and 5% CO 2. Linearized pWISG plasmid was electroporated into CHO cells (U =260 V,T =150 µs). G418 (Promega, Madison , WI, USA ) resistant clones of the CHO-transfected cells were analyzed directly for WEE1 and SCF/EGFP expression. Empty pIRES vec-tor and constructed pWIG plasmid (only EGFP was cloned
downstream of the IRES in pWI, data not shown) were
treated the same as pWISG as controls.
Immunohistochemistry analysis
CHO/pWISG and CHO/pIRES cells were fixed in ice-cold methanol, incubated with anti-WEE1 monoclonal antibody (mAb) (Santa Cruz Biotechnology, CA, USA ) 400-
fold diluted in BSA followed by incubation with PE conju-
gated goat-anti-mou IgG (Jingmei Biotech, Shenzhen,China). Nuclei were stained with Hoechst33342. Fluo-rescence was viewed under fluorescence microscope (Olympus, Tokyo, Japan).
Western blot analysis
107 CHO cells were washed in ice-cold phosphate-buffered saline (PBS) twice and lyd with 200 µl buffer A [10 mM HEPES, 10 mM KCl, 0.5% NP-40, 0.1 mM EDTA, protea inhibitor cocktail (Roche, Bal,Switzerland)]. Cytoplasmic extracts were recovered after
centrifugation (20,000 g for 5 min). Remaining pellets were
denatured with 25 µl buffer C (50 mM HEPES, 420 mM KCl, 0.1 mM EDTA, protea inhibitor cocktail). Pellets were maintained on ice for 30 min. Nuclear extracts were recovered after centrifugation (20,000 g for 20 min). Both fractions were analyzed by Western blot. The anti-WEE1mAb was ud at a 1:2000 dilution. Peroxida-labeled goat anti-mou IgG antibody (KPL, MD, USA) was ud at a 1:10,000 dilution. Proteins were detected using an ECL kit (Amersham Pharmacia, Freiburg, Germany).
mhSCF mRNA expression detection
RT-PCR was ud to detect mhSCF mRNA expression
in transfected CHO cells. Nested PCR were procesd
using the n (P3: 5'-GATAC
358CCGGGATGGATGT-
TTTG-3') and the antin (P2) primers.Flow cytometry analysis自学网
5×103 CD34+ cells were resuspended in supernatants of cultured transfectants, and incubated for 30 min at 37 °C in the dark. The cells were then rind twice with ice-cold PBS and resuspended in 500 µl PBS. Samples
Fig. 1 The bicistronic expression castte of pWISG
The elements of castte are as follows: P CMV , human cytomegalovirus immediate-early promoter/enhancer; IVS , intervening quence; ATG, initiation codon; TGA,
termination codon; IRES , internal ribosome entry site coding quence; mhSCF , extracellular domain of stem cell factor coding quence; EGFP , enhanced green
weak
fluorescence protein coding quence.
at Chine Academy of Medical Sciences on January 5, 2014
Feb., 2005 Ping LEI et al.: Bicistronic Plasmid Encoding Human WEE1 and SCF 109
were analyzed by FACSCalibur flow cytometer (BD Biosciences, CA, USA).
CD34+ cell paration
Umbilical cord blood (UCB) was obtained at the end of full-term deliveries after clamping and cutting of the cord by drainage of blood into sterile collection tubes contain-ing the anticoagulant citrate-pho
sphate dextro. Mononuclear cells (MNCs) were isolated from UCB using Ficoll Hypaque density centrifugation. The CD34+ MNC fraction was isolated with superparamagnetic microbeads lection using high-gradient magnetic field and midi-MACS columns (Miltenyi Biotec GmbH,Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. The CD34+ cells were cultured in IMDM (Gibco BRL) supplemented with 20% FBS plus 50 ng/ml TPO (PeproTech, London, UK), 50 ng/ml FL (PeproTech).
Anti-cisplatin damage studies
CD34+ cells were transfected with pIRES, pWISG, pWI and constructed pISG (only the mhSCF/EGFP fusion gene was cloned downstream of IRES in pIRES, without the WEE1 gene in upstream, data not shown), respectively. The percentage of damaged cells was identified using annexin V-FITC staining (BD) by flow cytometry (FCM) 48 h after adding cisplatin (Qilu Pharmaceutical Co., Ltd., Jinan, China) into media.
Results
Cloning and validation of bicistronic pWISG vector
The cDNA of the extracellular domain of SCF was ampli-fied from HepG2 cells by RT-PCR. The amplification re-action yielded fragments approximately 620 bp in length as expected. The digested PCR product was subcloned into pEGFP-N1 to produce pEGFP-N1/mhSCF and sub-jected to quence analysis. The cDNA quence and the predicted amino acid quence of mhSCF were in agree-ment with NM_000899 and AAK92485 in GenBank data-ba and published reports [7–9].
The structure of the bicistronic expression castte cloned into the pIRES vector is shown in Fig. 1. Restric-tion digestion mapping confirmed the prence of WEE1 and SCF/EGFP cDNAs in pWISG (data not shown).
Analysis for WEE1Hu protein
The location of the WEE1Hu protein was analyzed by immunohistochemistry using anti-WEE1 mAb. Nuclear fluorescence was obrved, consistent with nucleus-lo-calization of WEE1Hu [Fig. 2(B1)]. In contrast, no fluo-rescence was en with the control CHO cells [Fig. 2
(A1)]. Hoechst staining showed all of the nuclei [Fig. 2
(A2,B2)]. Expression of products were further analyzed
by Western blot. A specific protein band with a molecular
weight of about 50 kD emerged in the nuclear extract of pWISG transfectants, but neither in the cytoplasmic ex-
tract of the same transfectants nor in the total extract of
pIRES transfectants (Fig. 3).
Fig. 2 Immunofluorescence assay of WEE1Hu protein expresd in pWISG transfected CHO cells
fun怎么读CHO/pIRES (A) and CHO/pWISG (B) cells were analyzed using anti-WEE1 monoclonal antibody and Hoechst33342. (A1) and (B1) showed PE fluorescence.
(A2) and (B2) showed Hoechst fluorescence.
Fig. 3 Western blot analysis of the WEE1Hu protein in transfected CHO cells
The expression of WEE1Hu was confirmed in pWISG transfected CHO cells. 1,
nuclear extract from pWISG transfected cells; 2, cytoplasmic extract from pWISG
transfected cells; 3, total extract from empty pIRES transfected cells.
at Chine Academy of Medical Sciences on January 5, fordjournals/
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110 Acta Biochim Biophys Sin V ol. 37, No. 2RT-PCR detection for SCF mRNA expression Agar gel scanning showed there was a unique 440 bp product in pWISG transfected samples. Samples transfected with pWIG revealed no obvious 440 bp band (Fig. 4).approximately 56.27% of CD34+ cells cultured with the
supernatant of pWISG transfectants showed green fluorescence. However, the percentage was only 2.79%when cultured with the supernatant of pWIG transfectants and zero with the supernatant of empty pIRES vector
transfectants (Fig. 5). The results proved that the -
creted fusion protein retained its ability to bind to its re-
ceptor and the EGFP retained its own reading frame so
that it can be detected directly by FCM.
removal
Inhibition of cell damage by pWISG
To determine whether WEE1Hu and SCF expresd in
CD34+ cells can protect CD34+ cells from apoptosis, anti-
apoptotic function was estimated by annexin V-FITCsurvive什么意思
staining. After being expod to cisplatin for 48 h, 65.75%
of vehicle-transfected CD34+ cells underwent apoptosis,
while only 24.26% of pWISG-transfected CD34+ cells
were induced to be apoptotic. The percent of pISG-trans-
fected CD34+ cells and pWI-transfected CD34+ cells were
55.21% and 33.75%, respectively (Fig. 6).
Discussion
Hematopoietic stem cells are attractive targets for genewch
therapy becau of their capacity for lf-renewal and the
wide systemic distribution of their progeny. Sustained
expression of anti-apoptosis, anti-necrosis genes and/or
grow factors in the HSCs would provide novel and poten-
tially curative treatments for bone marrow suppression resulting from irradiation or chemotherapy treatment.Mitotic inhibitor WEE1 acts through the inhibitory phos-
mhSCF-EGFP fusion protein activity assay The supernatant of pWISG transfectants containing the putative mhSCF/EGFP fusion protein was mixed with CD34+ cells. FCM analysis for EGFP signal revealed that Fig. 4 mRNA expression of mhSCF using nested RT-PCR in
transfected CHO cells
Total RNA extracted from transfected CHO cells was subjected to RT-PCR analy-sis for mhSCF mRNA. M, DNA marker DL 2000; 1, RT-PCR product of pWISG
transfectants; 2, RT-PCR result of pWIG transfectants.Fig. 5 Flow cytometry analysis of the EGFP fluorescence
The supernatants of various transfected CHO cells were mixed with CD34+ cells for 0.5–2 h and then analyzed by flow cytometry for EGFP signal. The CD34+ cells binding with putative fusion protein showed green fluorescence and can be detected by FCM. The pIRES and pWIG plasmids rved as controls. The percentages of the subts are indicated. (A) The supernatant of pIRES transfected cells cocultured with CD34+ cells. (B) The supernatant of pWIG transfected cells cocultured with CD34+ cells. (C)The supernatant of pWISG transfected cells cocultured with CD34+
cells. One reprentative experiment of two is prented.
at Chine Academy of Medical Sciences on January 5, 2014
Feb., 2005 Ping LEI et al .: Bicistronic Plasmid Encoding Human WEE1 and SCF 111phorylation of Cdc2 on tyrosine 15 residue within the ATP-binding site of the Cdc2 catalytic subunit to delay mitosis in respon to DNA damage or a DNA replication block,thereby avoiding mitotic catastrophe [1–3,10]. Down-regulation of WEE1 kina can abrogate the G 2 check-point and induce apoptosis in human cervical carcinoma HeLa cells [11]. Our experiments also demonstrated that pCMV-WEE1-transfected NIT cells can resist the killing effect of cytotoxic T lymphocytes [12].SCF is a hematopoietic cytokine and cellular respon to SCF is initiated following its interaction with receptor c-kit, such as the migration of hematopoietic cells to their ultimate destinations during development. Constitutive production of SCF by marrow endothelial cells and fibro-blasts may be required for maintenance of normal basal hematopoiesis [5,6,13,14]. Accumulating findings show SCF could also be responsible for anti-apoptotic functions [14–17]. SCF is normally found in both soluble and trans-
membrane forms generated by alternative splicing of a proteolytic cleavage site in exon 6. Their extracellular N-terminal globular domain interacts with its receptor result-ing in the proliferation and differentiation of hematopoietic cells.We constructed a bicistronic vector encoding WEE1Hu and the extracellular domain of soluble SCF to investigate whether the introduction of WEE1Hu and SCF into HSCs could inhibit apoptosis induced by radio- and chemotherapy and promote HSC proliferation and differentiation.An increasing demand for more complex polycistronic vectors has arin in recent years to obtain complex gene transfer/therapy effects. In particular, this demand is stimu-lated by the hope of a more powerful effect from com-bined gene therapy than from single gene therapy in a pro-cess who parallels lie in the multi-drug combined thera-pies for cancer or AIDS. Strategies for co-expression of genes involve internal promoters, splicing, reinitiation, lf-processing peptides, proteolytic cleavable sites, fusion genes, IRES, and multiple transfection [18–21]. Internal ribosome entry is a eukaryotic translation initiation by a cap-independent recruitment of the 40S ribosomal sub-unit to internal mRNA quences. Applying IRES, veral proteins of interest can be synthesized from a single multicistronic mRNA, which overcomes the unparallel expression of the gene of the interest by other means.Furthermore, the property of certain IRES to be active in certain tissues and to be regulated under defined condi-tions can reprent an advantage for the targeting of transgene expression [21,22].
In the prent study, we detected the expression of WEE1Hu and SCF simultaneously in recombinant bicis-
tronic pWISG plasmid transfected CHO cells. Our experi-ment results confirmed that WEE1Hu protein was located in the nucleus by isolation of cytoplasmic and nuclear protein and cellular immunohistochemistry. The fusion protein of the extracellular domain of human soluble SCF and EGFP was creted into supernatant and retained its bifunction, the ability of binding to the SCF receptor and emitted green fluorescence. WEE1Hu and the fusion protein of SCF-EGFP were determined to be expresd in the parallel pattern. Our data showed that expresd WEE1Hu and SCF can protect the HSCs from apoptosis induced by cisplatin and fusion protein SCF-EGFP can bind to the SCF-receptor, promote HSC proliferation and emits green fluorescence. Our study implies that the strategy of using a bicistronic expression vector containing WEE1Hu and SCF-EGFP is more efficient than single gene therapy.
Fig. 6 Anti-drug damage analysis
Transfection with pWISG decread damage rate from exposure to cisplatin. Empty pIRES vector rved as control. (A) pIRES-transfected CD34+ cells. (B) pWISG-transfected CD34+ cells. (C) pISG-transfected CD34+ cells. (D) pWI-transfected CD34+
cells. The reprentative result of two experiments for each group is shown. at Chine Academy of Medical Sciences on January 5, 2014
112 Acta Biochim Biophys Sin V ol. 37, No. 2
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