Gene expression of growth signaling pathways is up-regulated in CD133-positive medulloblastoma cells

  • Authors:
    • Chunyu Gu
    • Naoki Yokota
    • Yun Gao
    • Junkoh Yamamoto
    • Tsutomu Tokuyama
    • Hiroki Namba
  • View Affiliations

  • Published online on: January 14, 2011     https://doi.org/10.3892/ol.2011.235
  • Pages: 357-361
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Abstract

Medulloblastoma (MB) is the most common malignant brain tumor in children. Cancer initiating cells (CICs) have been proposed to be involved in the development of brain tumors including MB. Prominin-1 antigen (CD133) is a candidate surface molecular marker for CICs. In the present study, CD133-positive cells were isolated from human Daoy MB cells and their gene expression was compared with that of control Daoy cells. DNA microarray analysis revealed that there were 398 up-regulated genes (>2-fold increase) and 318 down-regulated genes (<50% decrease) in the CD133-positive cell-enriched fractions. Up-regulated genes included neuregulin-1, cyclin D1, cyclin-dependent kinase 6, vascular endothelial growth factor, inhibin β A, promyelocytic leukemia gene, MYC, and hairy enhancer of split-1, which are components of growth signaling pathways. Molecular studies suggest that developmentally regulated signals important for stem cell maintenance are also involved in MB tumorigenesis. Moreover, these molecules can serve as novel targets for MB treatment.

Introduction

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and is thought to arise from precursor cells in the cerebellar granule cell lineage (1). MB patients are now divided into stratification groups according to age, degree of resection and disease dissemination, and are treated depending upon risk. Although the use of multidisciplinary approaches and stratification management of the disease have improved prognosis, 50% of patients, particularly in the high-risk group, experience disease recurrence, dissemination to the cerebrospinal fluid space, and/or a high incidence of sequelae (2).

The concept regarding the existence of cancer stem cells or cancer initiating cells (CICs) is currently a focal point. The hypothesis that cancerous cells originate from rare populations of CICs that are more tumorigenic than other cancer cells has gained increasing credence (3). CICs are thought to persist in tumors as a distinct population that can cause tumor recurrence and distant metastasis. The existence of CICs in MB has also been reported (4,5). Prominin-1 antigen (CD133) was identified in hematopoietic stem cells (6,7) and neuroepithelial stem cells (8) and has generally been used as a marker for CICs (9). Although some investigators assert that CD133 is not an adequate marker of CICs since both CD133-positive and -negative cells are able to initiate tumors (10), it is also true that CIC-like cells that exhibit self-renewal and multipotential properties are restricted in the CD133-positive cell fractions. In the present study, CD133-positive cells were isolated from the human Daoy MB cell line using magnetic-activated cell sorting (MACS) beads and the transcript profiles of CD133-positive Daoy MB cells were investigated using DNA microarray analysis in order to obtain a better understanding of the molecular properties of CICs involved in MB tumorigenesis.

Materials and methods

Cell culture

The human Daoy medulloblastoma (MB) cell line was purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, Inc., St. Louis, MO, USA) with 10% fetal bovine serum (FBS; Sigma-Aldrich, Inc.), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C under 5% CO2.

Flow cytometry

Cells were detached in phosphate-buffered saline (PBS) containing 0.25% trypsin and 0.02% EDTA for 3 min at 37°C under 5% CO2, and the reaction was stopped by adding complete medium (DMEM with 10% FBS). Following centrifugation at 1000 rpm for 5 min, the cells were washed and resuspended in bovine serum albumin (BSA)/PBS buffer (PBS with 0.1% BSA and 2 mM EDTA). Half of the cells were incubated with FcR blocking reagent (Miltenyi Biotec Inc., Auburn, CA, USA) and anti-CD133-PE (Miltenyi Biotec Inc.) for 10 min at 4°C, and the remaining cells were incubated with IgG-PE (BD Biosciences, San Jose, CA, USA) as controls. After washing, the cells were resuspended in BSA/PBS buffer and analyzed using the Beckman Coulter Epics XL system (Beckman Coulter, Inc., Chaska, MN, USA). The data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Cell sorting

CD133-positive Daoy cells were sorted using the CD133 cell isolation kit (Miltenyi Biotec Inc.). Briefly, cells were suspended in BSA/PBS buffer, incubated with FcR blocking reagent and CD133 microbeads (Miltenyi Biotec Inc.) for 30 min at 4°C. To determine the sorting efficiency, the cells were incubated with anti-CD133/2-PE for 10 min. Following washing and centrifugation, the cells were resuspended in BSA/PBS buffer, loaded onto a magnetic separation column (Miltenyi Biotec Inc.) and placed in a magnetic cell separator. The column was rinsed, and the magnetically labeled cells were flushed out with elution buffer and collected. These cells were used in the subsequent experiments.

DNA microarray analysis

Total RNAs were isolated from the CD133-positive Daoy cells (sorted and control) using TRIzol™ (Invitrogen, Carlsbad, CA, USA). Synthesis and labeling of cRNAs and hybridization of biotin-labeled cRNA probes to the Human Genome U133A 2.0 expression Chip arrays (Affymetrix, Santa Clara, CA, USA) were performed according to the manufacturer's protocol. The imaging screens were scanned and analyzed using the Affymetrix Microarray Suite and GeneSpring GX (Agilent Technologies, Santa Clara, CA, USA).

Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analyses

Total RNAs were prepared and used as templates for cDNA synthesis with random hexa-nucleotide primers and SuperScript reverse transcriptase II (Invitrogen). Real-time PCR analyses were performed using a QuantiTect SYBR-Green PCR kit (Takara, Kyoto, Japan) and a LightCycler System (Roche, Basel, Switzerland). The PCR primer sequences were determined using WWW primer tool, Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) (Table I). The transcript abundance of the genes of interest was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal standard. At least 3 independent analyses were performed for each sample and for each gene.

Table I

RT-PCR primer sequences of the genes of interest.

Table I

RT-PCR primer sequences of the genes of interest.

Gene symbolGenebankSenseAntisense
NRG1NM013959 TTGGTGCTGCTTTCTTGTTG CGGAGCCTCACACACCTATT
CCND1BC000076 TCCTCTCCAAAATGCCAGAG TGAGGCGGTAGTAGGACAGG
CDK6NM001259 AGGGTGCAGTCAAAACAACC TCCCATCCACTTCAAAGGAG
VEGFAF091352 TGCAGATTATGCGGATCAAA GCGAGTCTGTGTTTTTGCAG
INHBAM1343 AGACGCTGCACTTCGAGATT CCCTTTAAGCCCACTTCCTC
JAG1U73936 AGCTGGCTTACACTGGCAAT AAGTGGGAGCTCAAAGACCA
MYCNM002467 CTCCTGGCAAAAGGTCAGAG TCGGTTGTTGCTGATCTGTC
HES1NM005524.2 CTCTCTTCCCTCCGGACTCT AGGCGCAATCCAATATGAAC
PMLAF230411 GCAGCAGTGAGTCCAGTGA GCTCTGCCTGCACTTCTTT
NFASCNM015090.2 TGCCTTGCTTTTGAGGAGAT GGCTGTGGTCAGGGAAACTA
APOENM000041 CCAATCACAGGCAGGAAGAT AGCGCAGGTAATCCCAAAAG
ASTNAB0006627 ACAACACCCTCCTGGATCTG AAGGAGTCCATTGCACCAAC
BMP2NM001200 GGAGAATGCCCTTTTCCTCT ACAACCCTCCACAACCATGT
NEFLNM006158.2 TCTGTTTGCTTGCAGAGTGG GCTAACCACCGAAGGTTCAA
MAP2U89330 AAGAAGGTCGCCATCATACG GGCGGATGTTCTTCAGAGAG
GAPDH TGCACCACCAACTGCTTAG GAGGCAGGGATGATGTTC

Results

CD133-positive Daoy MB cells were highly enriched by MACS

Flow cytometry showed that 3–5% of Daoy cells expressed prominin-1 antigen (CD133) and CD133/2 antigens. After MACS was applied, the CD133-positive cells were highly enriched (>60%). These cells were then used in the DNA microarray gene expression analyses.

Transcript analysis in CD133-positive MB cells

Transcript analysis using DNA microarrays was performed, and the acquired data were filtered according to the gene expression level. In comparison with the control Daoy cells, the CD133-positive cell-enriched fractions exhibited a >2-fold increase in the expression of 398 genes, and a <50% decrease in the expression of 318 genes. A number of molecules involved in the growth signaling pathways, which play important roles both in MB oncogenesis and stem cell proliferation, were up-regulated in the CD133-positive cell-enriched fractions. These molecules included neuregulin-1 (NRG1; which showed a 6.818-fold increase), cyclin D1 (CCND1; 5.636), cyclin-dependent kinase 6 (CDK6; 3.564), vascular endothelial growth factor (VEGF; 3.186), inhibin β A (INHBA; 3.115), Jagged 1 (JAG1; 2.702), promyelocytic leukemia gene (PML; 2.538), MYC (2.479), and hairy enhancer of split-1 (HES1; 2.078) (Table II). On the other hand, neural differentiation markers or developmentally regulated genes, expressed in the granule cell lineage, such as neurofascin (NFASC; 0.0608), apolipoprotein E (APOE; 0.296), astrotactin (ASTN; 0.392), neurofilament light polypeptide 68 kDa (NEFL; 0.418), and microtubule-associated protein 2 (MAP2; 0.49) were down-regulated (Table II). Semi-quantitative RT-PCR analyses were then performed in the selected genes (up-regulated genes, Fig. 1A; down-regulated genes, Fig. 1B) and the gene expression changes were confirmed to be significant.

Table II

Gene changes in the CD133-positive Daoy cells.

Table II

Gene changes in the CD133-positive Daoy cells.

SymbolGenebankMapFold changeGene name
Up-regulated genes
 RGS16U948291q25-q3110.87Regulator of G-protein signaling 16
NRG1NM_0139598p21-p126.818Neuregulin 1; a ligand for the NEU/ERBB2
CCND1 BC00007611q135.636Cyclin D1
 JUNBC0026461p32-p315.161V-jun sarcoma virus 17 oncogene homolog (avian)
 CASP2BC0024277q34-q355.081Caspase 2, apoptosis-related cysteine peptidase
 EGR1NM_0019645q31.13.963Early growth response 1
 METAA0051417q313.886Met proto-oncogene (hepatocyte growth factor receptor)
 Cep290AF31788712q21.333.883Centrosome protein cep290
CDK6 NM_001259 7q21-q223.564Cyclin-dependent kinase 6
 MAXNM_00238214q233.531MYC associated factor X
 DKK1NM_01224210q11.23.277Dickkopf homolog 1 (Xenopus laevis)
VEGF AF0913526p123.186Vascular endothelial growth factor
INHBAM13436 7p15-p133.115Inhibin, β A (activin A, activin AB α polypeptide)
 PYGO1AL04992515q21.13.104Pygopus homolog 1 (Drosophila)
JAG1U73936 20p12.1-p11.232.702Jagged 1 (Alagille syndrome)
 HDAC9NM_0147077p21.12.651Histone deacetylase 9
 KHSRPAI93330119p13.32.643KH-type splicing regulatory protein (FUSE binding protein 2)
 NPATU5885211q22-q232.564Nuclear protein, ataxia-telangiectasia locus
 GADD45BNM_01567519p13.32.563Growth arrest and DNA-damage-inducible, β
PML AF23041115q222.538Promyelocytic leukemia
 GREM1NM_01337215q13-q152.508Gremlin 1, cysteine knot superfamily, homolog
MYC NM_002467 8q24.12-q24.132.479
 CCNT1NM_00124012pter-qter2.41Cyclin T1
 TGFBR1NM_0046129q222.408Transforming growth factor, β receptor I
 EGFRU950897p122.299Epidermal growth factor receptor
 CCNE2AF1128578q22.12.162Cyclin E2
 SMAD5AF0106015q312.125SMAD, mothers against DPP homolog 5 (Drosophila)
HES1 BE973687 3q28-q292.078Hairy and enhancer of split 1 (Drosophila)
 SMAD3NM_00590215q21-q222.016SMAD, mothers against DPP homolog 3 (Drosophila)
Down-regulated genes
NFASC AI8217770.0608Neurofascin homolog (chicken)
 IGF1AI97249612q22-q230.166Insulin-like growth factor 1 (somatomedin C)
 IGFBP5AW0075322q33-q360.175Insulin-like growth factor binding protein 5
 SEMA3ENM_0124317q21.110.197Semaphorin 3E
APOE NM_00004119q13.20.296Apolipoprotein E
 BBPAA0129171p32.10.356TM2 domain containing 1
 VCAM1NM_0010781p32-p310.359Vascular cell adhesion molecule 1
 SLIT3AB0115385q350.328Slit homolog 3 (Drosophila)
 RARRES2BC0000697q36.10.365Retinoic acid receptor responder (tazarotene induced) 2
ASTN AB0066271q25.20.392 Astrotactin
BMP2 AA58304420p120.397Bone morphogenetic protein 2
 TNCBF4348469q330.404Tenascin C (hexabrachion)
 UNC5BAA12788510q22.20.406Unc-5 homolog B (C. elegans)
NEFL AL5374578p210.418Neurofilament, light polypeptide 68 kDa
 RBP1NM_0028993q230.448Retinol binding protein 1, cellular
 CDH11AU1443780.459Cadherin 11, type 2, OB-cadherin (osteoblast)
 RAI16NM_0227498p21.30.461Retinoic acid induced 16
 CASP4AL05039111q22.2-q22.30.48Caspase 4, apoptosis-related cysteine peptidase
MAP2U89330 2q34-q350.49 Microtubule-associated protein 2

[i] The genes of interest are indicated in boldface type.

Discussion

In the present study, we first isolated CD133-positive cells in the human Daoy medulloblastoma (MB) cell line. The percentage of CD133-positive cells was approximately 3–5%, which was in accordance with previous studies (0.5–10%) (11,12). After MACS was applied, the percentage of CD133-positive cells was noted to be greater than 60%. These enriched cell fractions were subsequently subjected to transcript analysis using DNA microarrays.

Transcript analysis using DNA microarrays identified various molecules that were components of the growth signaling pathways, which play important roles both in MB oncogenesis and stem cell proliferation. The genes which exhibited up-regulated expression included the activator of MAP kinase signal (RGS16), a ligand of ERBB (EGF signal component; NRG1), Wnt signal targets CCND1 and c-myc, a ligand of Notch signal (JAG1), and its target (HES1) (1315) (Table II). c-myc is known to play a key role in stem cell self-renewal and was used to produce induced pluripotent stem cells (16). The Wnt and Notch pathways are involved in the maintenance of stem cell properties and in MB oncogenesis (1719). The remaining up-regulated genes included INHBA, an inhibitor of differentiation factors, such as activin and TGF β, and VEGF which plays a role in the neovascularization of tumors (20,21). These genes may be involved in tumor recurrence or distant metastasis.

In contrast, the genes whose expression decreased to less than 50% included the neural markers (MAP2 and NEFL) (22,23), developmentally regulated genes in the cerebellar granule cell lineage (NFASC, UNC5B, ASTAN, SLIT3 and APOE) (2427), and molecules involved in retinoic acid-induced apoptosis in neuroblastoma (RBP1, BMP2, RARRES2 and CASP4) (28,29). Down-regulation of these genes may result in the inhibition of differentiation and maintenance of undifferentiated properties of CICs or may contribute to the inhibition of cell death, thereby providing infertility to CICs.

An understanding of the molecular pathway involved in MB oncogenesis has been advanced by analyses of the Turcot- and Gorlin-inherited syndromes which are associated with the development of MB. The Wnt and sonic hedgehog (SHH) signal pathways are involved in MB oncogenesis in the Turcot and Gorlin syndromes, respectively (3033). In addition, the Notch, epidermal growth factor receptor ERBB, and platelet-derived growth factor (PDGF) signaling pathways are involved in MB oncogenesis or prognosis (3337). These pathways play crucial roles in the proliferation and/or differentiation of the cerebellar granule cell lineage where MB originates. Furthermore, molecular studies have shown that developmentally regulated signals, such as Wnt, SHH and Notch, play important roles in self-renewal, proliferation and/or the multipotency of stem cells, and are also involved in MB oncogenesis (1719). These molecular studies and the results of the present study indicate that further understanding of the molecular properties and fundamental signaling pathways of CICs involved in MB oncogenesis may lead to the development of new, more effective, and less toxic treatment modalities for MB, thereby improving the quality of life of children with MB.

Acknowledgements

We express sincere appreciation to Professor Y. Koide, Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Professor T. Nagata, Dr S. Seto and Dr M. Uchijima and other members of Professor Koide's Laboratory for helpful advice, technical assistance and valuable discussions. This study was supported by a fund from the Japanese Ministry of Education, Culture, Sports, Science and Technology (no. 17501509).

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Gu C, Yokota N, Gao Y, Yamamoto J, Tokuyama T and Namba H: Gene expression of growth signaling pathways is up-regulated in CD133-positive medulloblastoma cells. Oncol Lett 2: 357-361, 2011.
APA
Gu, C., Yokota, N., Gao, Y., Yamamoto, J., Tokuyama, T., & Namba, H. (2011). Gene expression of growth signaling pathways is up-regulated in CD133-positive medulloblastoma cells. Oncology Letters, 2, 357-361. https://doi.org/10.3892/ol.2011.235
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Gu, C., Yokota, N., Gao, Y., Yamamoto, J., Tokuyama, T., Namba, H."Gene expression of growth signaling pathways is up-regulated in CD133-positive medulloblastoma cells". Oncology Letters 2.2 (2011): 357-361.
Chicago
Gu, C., Yokota, N., Gao, Y., Yamamoto, J., Tokuyama, T., Namba, H."Gene expression of growth signaling pathways is up-regulated in CD133-positive medulloblastoma cells". Oncology Letters 2, no. 2 (2011): 357-361. https://doi.org/10.3892/ol.2011.235