Stem cells are defined that divides to generate on daughter cell that is a stem cell and another daughter cell that produces differentiated descendants (Weissman et al, 2001). It termed as self renewal and differentiation. Self renewal can be defined as making a complete phenotype of stem cells through symmetric division like mitosis and unlimitedly divided.
Most somatic cells cultured in vitro display a finite number of population doublings and this can be contrasted with the seemingly unlimited proliferation capacity of stem cells in culture (Schofield, 1983). Differentiation is defined by acquisition of cell type specific morphological, phenotypic and functional features (Conley et al, 2004; Cryz et al, 2001, Baksh et al, 2004). In process of development, fertilized egg divided to multiple cells and became blastocyst that will be embryo consisted with multicellular organism. Differentiation means that development of zygote. In this procedure, some cells have the potential to form most or all type of specialized cells and this phenomenon called pluripotency (Mitalipov et al, 2009). Stem cells broadly consist with embryonic stem cell and adult stem cell. Embryonic stem cells (ESCs) isolated from the inner cell mass of blastocyst and have self renewal and pluripotency (Conley et al, 2004). Adult stem cells (ASCs) isolated from various adult tissues and have self renewal ability (Clarke et al, 2000). Discriminated with ESCs, ASCs have multipotency that differentiates into a number of cells, but only those of a closely related family of cells. Neural stem cells (NSCs) are one of ASCs. NSCs have the capacity to self renew and produce precursors that will differentiate to neurons and glia cells (astrocyte
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and oligodendrocyte) (Mckay et al, 1997). NSCs isolated from the striatal tissues including the subventricular zone and hippocampus (Reynolds et al, 2005). In addition, NSCs have migration ability to damaged tissues such ac Parkinson’s disease, stroke and spinal cord.
Many previous experiments reported that NSC migrates to abnormal tissues and recovery. In Parkinson’s disease model, caused by selective doparminergic neuron cell death in substantia nigra (SN), transplanted NSCs recovered MPTP induced neuronal cell death in SN region (Imitora et al, 2004). In ischemic stroke model and spinal cord injury model, injected NSCs moved to damage region and differentiated to brain component cells such as astrocyte or oligodenrocyte (Keirstead et al, 2001). Additionally, NSC transplanted animals showed behavior improvement and damaged tissues size reduction (Daadi et al, 2010; Teng et al, 2002). NSCs migration ability observed in animal brain tumor model. Transplantation of NSC into the animal cerebral hemisphere contralateral to the hemisphere containing an implanted glioma, lead to migration of those transplanted cells over long distance to the tumor and infiltrating tumor cell. Gliomas rise by various origins included genomic instability, oncogene expression, abnormal cell cycle regulation and unknown factors (Reuss et al, 2009; Moor et al, 2009). Malignant tumors have very aggressive infiltration behavior to near normal brain parenchyma and caused reduce patients survival period (Fine et al,1993;
Surawicz et al, 1998). Despite cancer therapeutic methods, surgery, chemotherapy, radiotherapy, was improved, the results was not satisfied. Because of tumor surrounding brain structure, effecting complete resection is difficult. So, necessitate the use of radiochemotherapy for effective tumor control. The use of chemotherapy is limited by permeation of drug to brain tissue that usually blocked with blood brain barrier and
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radiotherapy is restricted by its own toxicity on normal brain tissue. Overcome to these restriction, idea of using stem cell for cancer therapy as therapeutic reagents deliver with stem cell tropism property for tumor was stared from Aboody et al that showed transplanted neural stem cell into glioma bearing animals migrated to main tumor mass and invading gliomacell into near normal parenchyma (Aboody et al, 2000). After this report, many groups tried to find more efficient factor for cancer therapy using genetically modified gene bearing stem cells, PEX (Kim et al, 2005), IFN-b(Ito et al, 2009), S-TRAIL (Menon et al, 2009) CD with 5FC (Shimato et al, 2007) which have cancer growth inhibit factor. Those anticancer factors efficiently reduced tumor size in animal glioma model. Each gene encoding NSCs transplanted to contralaterally glioma cell bearing animal models. Few days later, transplanted NSCs present their anticancer effect and significantly reduced tumor size with expanding animal life span. It is mean that NSC could be efficient deliver vehicle of anticancer factors. Even though above trials have therapeutic effect in animal model, brain tumor, stroke and various brain diseases (Aboody, 2008; Miljan, 2009; Nandoe, 2009; Salehi, 2009 Hwang, 2010), some is known about signal transduction mechanisms of NSC tropism.
Stromal cell derived factor-1(SDF-1)/CXCR4 and vascular endothelial growth factor (VEGF)/ VEGFR systems were famous for stem cell migration induced factor (Bao, 2006;
Horvath, 2004). Increased NSC migration rate by SDF-1 or tumor cultured media compare normal condition was reduced in SDF-1 monoclonal antibody or CXCR4 blocking antibody treated condition (Imitola et al, 2004; Ehtesham et al, 2004). Different type of stem cell, mesenchymal stem cells, has same result (Son et al, 2006). In VEGF treated condition, NSC migration ability was increased. VEGF infused to one rat hemisphere cortex then
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transplanted NSC to counterpart cortex migrated to VEFG infusion site (Schmidt et al, 2005).
Additionally, Neuroinflammation derived chemokines can role as NSC attract molecule. In hippocampal slice culture, neural progenitor cells move from transplanted site to inflammatory stimulus mediated inflammation site that increased the chemokines and cytokines expression. Inflammation induced NSCs migration was inhibited by monocyte chemoattractant protein-1 (MCP-1) neutralizing antibody (Belmadani et al, 2006). In addition, HGF, TGF, PTN and PDGF induced NSC chemotactic migration (Heese et al, 2005).
Mentioned factors of above is activate intracellular signaling pathway integrate to each receptor. Most broadly used factor is FAK, cytoplasmic kinase, which involved in extracellular matrix and integrin mediating signaling pathway (Kornberg et al, 1998). FAK signaling pathway have important role in tumor invasion through regulation of cell migration (Meng et al, 2009). Actually, FAK kinase activation was upregulated in glioma cell line and tissues. Activated FAK transmit signals through PI3K that promote phosphorylation of downstream targets such as AKT in many kinds of glioma cells (Zheng et al, 2000). The migration character of glioma cells and NSCs has similar behavior properties. Glioma secreted various chemokines and growth factors that induce NSC tropism for glioma microenvironment. VEGF, highly expressed in glioma and crucial role in angiogenesis for tumor growth, bind to VEGFR that controlled FAK and PI3K activation. Both kinase regulated cell migration through distinct pathway by promoting the dynamic regulation of focal adhesion and actin structures (Giles, 2001). Some of reports elucidate that the phosphoinositide 3 kinase (PI3K) signaling or ERK and SAPK/JNK signal (Honeth et al,
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2006) play important role in stem cell migration (Kendall et al, 2008; Zheng et al, 2007).
Originally, neural migration critically controlled for positioning of neurons during brain cortex development (Kawauchi et al, 2008). Process of neuronal migration was consisted with neurite outgrowth and translocation of nucleus (Tasi et al, 2005). At the first, leading process is extended to want direction followed nucleus and cell body are moved using cytoplasmic dynein and microtubule rearrangement. In this procedure, specifically nucleus translocation, DCX and Lis1 with Nudle1 was crucially play role for nucleus and centrosomal coupling (Neithammer et al, 2000). These proteins disruptions lead to break of microtubules bridging between centrosome and nucleus and controlled by CDK5 signaling pathway. Abnormality of CDK5 and downstream targets is related with neurodegeneration.
This neuronal migration regulated the affect of chemoattractive and chemorepellent cues for properly neurogenesis.
Endogenous NSCs in the adult brain, it is known that their self recovery activity for the various injuries is inadequate or ineffective. Decreasing of proliferation ability, particularly in the adult brain, may because of the limited number and restricted location of endogenous NSC. NSCs expanded in vitro culture and transplanted into diverse damaged regions for overcome those limitations such as inadequate number and regions. Exogenous NSC may be inhibited in surviving or repair behavior by endogenous microenvironment. However, in above experiments, expanded NSC in vitro was well survived and showed tropism for damaged tissues when they introduced to abnormality having animals. Migrated NSC present several therapeutic effect include improvement of behavior. For these good effective result, repetition of NSC transplantation was needed and should optimize that most effectible
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with minimum number of NSCs. NSCs necessary critical controlled. However, the information about NSC migration mechanism is little known and not clears.
Many previous reports said that tumor or damaged tissues derived factors regulate NSC migration that could be used delivery vehicle or therapeutic factors through various signal transduction mechanisms. However, there are not believable key molecules and mechanisms that specialized cancer therapeutic strategy using optimized stem cells.
I hypothesize that commonly expressed molecules in different kind of tumors, especially malignant tumors were existed. It could main key-molecule that induces NSC tropism for tumor powerfully with unique signal transduction pathway. Beside, these molecules give positive effect, suggest the direction and promote motility, to cancer curable gene carrying NSC for antitumor function.
For investigate these ideas, I indentified tumor specific expressed gene using microarray in three different tumors and confirmed existence of malignant tumor specific gene with real time PCR in many kinds of tumors that classified as WHO grades. Because of most tumor produced factors involved in NSC migration, identified gene tested chmoattractive ability to NSCs. Whether this gene became key molecule in NSC tropism for tumor, it compared with well known factor of NSC migration inducer using in vitro assay. Additionally, chemoattractive property of this gene was double check in vivo condition. In tropism procedure of NSC, whether the signaling pathway of glioma cell migration and neuronal migration were activated, I checked. Finally, could identified gene increase stem cell based cancer therapy, apply of anticancer gene encoding NSC with systemic treated prodrug, in vivo bystander effect assay carried out in rat glioma model.
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Ⅱ. Materials and Methods
1. Cell culture and establishment of stable cell lines
Rat glioma cells, C6, and human neural stem cells, F3 and mouse fibroblast cell line, NIH3T3, were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum(FBS), 10ug/ml penicillin-streptomycin (Gibco, NY, USA) and incubated at 37oC in an incubator of 5% CO2/95% air.
2. In vitro migration
Cultured human neural stem cells were detached with 0.05% trypsin, washed in DMEM medium, resuspended in DMEM medium, and 5 x 104 cells seeded into the upper chambers (Costar Transwell) in 200μl DMEM with 10%FBS. 1day later, The lower chamber was filled with POSTN 20㎍/㎖or VEGF 20 ng/㎖ contained DMEM and the 10%FBS-DMEM medium was used as a negative control. To measure protein inhibitor effect, the lower chamber was filled with combined solution that composite with POSTN (R&D), VEGF, 20nM PD98059 and 20nM Roscovitine (Calbiochem). After 12h the insert was removed from the transwell. Remained Cells in the upper chamber were scraped off with cotton wool and stained with hematoxylin and count stained with eosin. And count number of directly movement cells that across an 8-μm-pore polycarbonate membrane towards lower chamber.
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To investigated ERK and DCX affection to migrating cell, we performed transwell membrane immunostain with ERK and DCX antibodies (Cell signaling technology. Danver, MA). Membrane were washed with PBS and then blocked with 0.5% triton X-100 and 5%
bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min followed two times washed with 0.5%BSA in PBS for 15min each. And membrane incubated with goat polyclonal human DCX antibody at a 1:500 and rabbit polyclonal human ERK antibody at a 1:1000 dilution overnight at 4℃. Then membrane washed with PBS for 15min 2 times.
Subsequent membrane incubated with biotin conjugated anti-rabbit antibody for 1 hour at room temperature followed wash 2times with PBS and complexes with avidin-biotin (Vector laboratories, Burlingame, CA). Then 3 times washed with PBS and detected using a diaminbenzidine peroxidase (DAB) kit (Vector laboratories, CA).
3. Collection of normal brain and brain tumor tissue
Patient tumor and normal brain samples were give from M.D. Cho in Ajou university hospita. Specimens were snap soak in RNAlater and stored -70 oC until use. All of the patients signed consent for collection and analysis of samples.
4. RNA isolation and application to Affymetrix GeneChip
Total RNA samples were prepared from 12 human brain tumors and 5 normal brains with the TRIzol reagent (Invitrogen) according to the manufacturer’s directions. Probe synthesis
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and hybridization of human U 133 Plus 2.0 GeneChip Oligo Microarrays (Affymdetrix, Santa Clara, CA, USA) were performed in accordance with the manufacture instructions.
Gene expression data (CHP file of Affymetrix Microarray Suite 5.0 software) were normalized to global target intensity.
5. Real-time quantitative RT–PCR
Total RNA (50ng) was used to generate cDNA using the Taqman Reverse Reagents (Applied Biosystems, Foster City, CA, USA). Taqman Universal PCR Master Mix and Taqman Gene Expression Assays, which contain the primers for candidate genes (CCL4, CXCL10,CYR61, FN1, GLIPR1, GPX3, HLA-DQB1, POSTN, PROS1, SFRP4, TIMP1, TIMP2, TNC, TNFRSF1A, TNFSF13B and GAPDH as internal control, Applied Biosystems, Foster City, California), were performed for detecting RT–PCR products. The PCR cycling conditions were performed for all of the samples, as follows: 2 min at 501C for incubation; 10 min at 95 oC for AmpliTaq amplification and 40 cycles for the melting (95 oC for 15sec) and annealing/extension (60 oC for 1 min) steps. PCR reactions for each template were performed in triplicate in one 96-well dish per gene-specific primer pair tested. The comparative CT method was used to quantification of the gene expression.
6. Western Blotting Analysis.
Cells were washed with phosphate-buffered saline (PBS), and directly lysed in RIPA
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buffer (0.5% sodiumdeoxycholate, 0.1% sodiumdodecyl sulfate, 1% NP-40 and PBS) with PMSF and proteases inhibitors. Protein concentration was determined by Bio-Rad DC assay (Bio-Rad, Hercules, CA, USA). The samples were adjusted to equal protein concentrations, 30㎍ of each protein sample was separated on an SDS-PAGE gel. The protein samples were transferred to immobilon-P membrane (Millipore Corporation, MA, USA). For protein detection, the membrane was incubated in 5% skim milk in TBS-T for 30min at room temperature followed washed three times with TBS-T for 5 min each. The membrane was then incubated with ERK, pERK, DCX (1:1000 dilution, Cell signaling Technology), pDCX (1:500 dilution, Cell signaling Technology), FAK, pFAK, PAK and pPAK (1:1000 dilution, Epitomics) primary antibody in 5% BSA-TBS-T at 4 oC for overnight. The membrane was then washed three times in TBS-T, 15 min each, and incubated with the secondary anti-rabbit antibody, 1:2000 dilutions (Zymed, CA, USA) for 1 h at room temperature. The membrane was washed three times in TBS-T, and antibody-bound protein was detected by adding ECL Western Blotting Detection reagent (Amersham Pharmacia Biotechnology, buckinghamahire, UK) for 1 min and exposing the membrane to Kodak X-ray film.
7. Reverse transcription - PCR
First-strand cDNA was generated by SuperscriptⅡ(Invitrogen, carlsbad, CA) and 12-18mer oligo dT with 2mg of total RNA that was isolated using TRIzol reagent (Invitrogen) in F3 neural stem cell. The reaction mixture was incubated at 42℃ for 50min followed by incubation at 72℃ for 15min. 150ng of cDNA was then used as template in a 50㎕ PCR
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containing 5ml 10X PCR buffer, 1.5mM MgCl2, 0.2mM deoxyribonucleoside-triphosphate, 50pmol primer and 1U of Tag DAN polymerase (Invirogen). The PCR conditions were as follows: 25~30 cycles at 94℃ for 45 seconds, 55℃ for 45 seconds, 72℃ 45 seconds (CD:
60℃ for 60 seconds, 72℃ 90 seconds) and a final extension for 10minutes at 72℃. The primer sequences for integrin genes were as follows: for human integrin alpha V, 5’-ACT GGG AGC ACA AGG AGA ACC-3’ and 5’-CCG CTT AGT GAT GAG ATG GTC-3’; and for human integrin beta1, 5’-CTG CAA GAA CGG GGT GAA TG-3’ and 5’-CAC AAT GTC TAC CAA GCC C-3’; and for integrin beat 2, 5’-CAA GCT GGC TGA AAA CAA CA-3’ and 5’-ACT GCT CCT GGA TGC ACT CT-3’; and for integrin beta3, 5’-AGA TGC GAA AGC TCA CCA GT-3’ and 5’-CCG TCA TTA GGC TGG ACA AT-3’; and for integrin beta4 5’-GCC TTC ACT TTG AGC ACT CC-3’ and 5’-CTG CTG TAC TCG CTT TGC AG-3’; and for integrin beta5, 5’-AGC AGC TTC CAT GTC CTG AG-3’ and 5’-GAA GTT GCT GGT GAG CTT CC-3’; and for integrin beta6, 5’-GAC TCC GGA AAC ATT CTC CA-3’ and 5’ CTG ACA GTC GCA GTT GCA TT-3’; and for integrin beta 7, 5’-AGC AAT GGC CTC TAC AGT CGC AGC-3’ and 5’-GCT TGG AGA GAA ACC CAG AAA GTC-3’; and for integrin beta 8, TTC ATC ATT TTC ATA GTT ACA TTC-3’ and CAT TAA GTG TTT AAA AAT CTT TTT-3’, and for cytosine diaminase (CD), 5’-GAGTCACCGCCAGCCACACCACGGC-3’ AND 5’-GTTTGTAATCGATGGCTTCT GGCTGC-3’
8. Immunohistochemistry
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Human brain tumor and normal brain tissues were fixed with ice-cold 4%
paraformaldehyde in 0.1M phosphate buffer. After 4hr, the tissue transfer to 30 % sucrose in 0.1 M phosphate buffer at 4°C for overnight. Then tissue were frozen with OCT compound and sectioned at 30mm on a cryostat. The slides were washed with PBS 3 times and blocked with 0.5% triton X-100 and 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 minute. After then, slices washed with 0.5% BSA in PBS for 15min each followed incubated with rabbit polyclonal human Periostin antibody (1:500, abcam, Cambridge, UK) for overnight at 4℃. Slices were washed three times with PBS, 15 min each, and incubated in the secondary anti rabbit antibody (1:1000 dilutions (Vector laboratories, CA, USA) contained PBS for 1h at room temperature. After that, periostin was detected with diaminbenzidine peroxidase (DAB) kit (Vector laboratories, CA) and counterstained with hematoxylin.
9. Construct of Periostin overexpressing cell line
pCXbsr and pCL Ampho vector, kindly provide by Dr Inoue in Shiga university of medical science of japan, were transfected to 293T cells using the lipofectamin and plus agent according to the manufacture’s protocol (invitrogen, Carlsbad, CA). 2 days later, blasticidin S 5g/ml (invitrogen, Japan) treated to transfected 293T cells for a week and changed new media except for blasticidin S. 3 days after, cultured 293T cell media were transferred to NIH3T3 cells and incubated for 2 days. Then, blasticidin S contained media
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was added and selected for 1-2 week. Formed colonies were cultured to 96well plate as one cell to one well and each cells used as periostin overexpressed cell lines (p-NIH3T3).
10. In vivo migration
Three micro-liter Hanks’ balanced salt solution (HBSS) or Dycycle green labeled p-NIH3T3 cells (1x106 cells) had stereotaxically injected into the right striatum. The coordinates, anterior-posterior (AP) measured from bregma, lateral (ML) from the midline, and depth (DV) from dura, were as follow (in ㎜): AP +0.4; ML -2.3; DV -4.5. Five day after, each group was stereotaxically injected with DiI labeled 1x106 NSCs into same hemisphere cortex that coordinates, anterior-posterior (AP) measured from bregma, lateral (ML) from the midline, and depth (DV) from dura, were as follow (in ㎜): AP +0.4; ML -2.3; DV -2.0. Cells and HBSS injected at a rate of 0.2ul/min using 26-gauge Hamilton syringe (Hamilton, Nevada, USA) attached to an automated microinjector (KD scientific INC, MA, USA). After transplantation, the needle was left in place for an additional 15min before being slowly retracted. 2-4weeks later, rats were sacrificed and collected rat brain fixed with ice-cold 4 % paraformaldehyde in 0.1M phosphate buffer. After 4hr, brains sink with 30 % sucrose in 0.1 M phosphate buffer at 4°C for overnight. Then brains frozen with OCT compound and sectioned at 30mm with cryostat. Slices were mounted with Vectashield® Hard Set™ mounting medium (Vector, CA, USA), and were observed with an Olympus IX71 confocal laser scanning microscope (Olympus, Tokyo, Japan).
14 11. Analysis of CD enzyme activity
F3-CD Cells (2X105 cells) were inoculated each well in 12-well plate and incubated for 48 hours with 1 mM 5-FC in 1ml medium. Incubated medium (50ml) was extracted with 500µl ethyl acetate:isopropanol:acetic acid (84:15:1 [v/v/v]), and the organic fraction was resuspended with 500µl H2O:methanol (4:1 [v/v]). HPLC carried out using a Kromasil 100-5C-18 column (Kromasil, Bohus, Sweden) and detected at UV 270nm. 5-FC and 5-FU were eluted isocratically at a flow rate of 1 ml/min with an isocratic mobile phase consisting of 40mM KH2PO4, adjusted to pH 7.0 with 10 % KOH. Retention times were 3.4 minutes for 5-FC, 3.9 minutes for 5-FU and 8.9 minutes for 5-bromouracil (Sigma), an internal standard.
Quantification of 5-FC and 5-FU was normalized with 5-bromouracil.
Fifty micrograms proteins of cell lysates were incubated in the presence of 1mM 5-FC at 37℃ for 8 hours for enzyme activity. The mixtures were extracted and analyzed by HPLC as described above.
12. In vitro bystander effect assay
To observe bystander effect of p-NIH3T3 cell by 5-FC treated F3-CD, Total 1x104
To observe bystander effect of p-NIH3T3 cell by 5-FC treated F3-CD, Total 1x104