Tumor specimens were obtained with informed consent from a 37-year-old female patient who underwent initial surgical removal in January 1999 and subsequent recurrent resections of a right temporal tumor July 1999 and February 2001 at the 2nd Affiliated Hospital of Soochow University (Suzhou, China) and did not receive chemotherapy and radiation between these stages of progression. The tumor was diagnosed as ganglioglioma (WHO grade I), anaplastic astrocytoma (WHO grade III) and glioblastoma (WHO grade IV) on paraffin embedded sections (Figure ). Normal brain tissues were obtained from the same patient when she was undergoing partial removal of the right occipital lobe for cerebral decompression. Part of the fresh tissue was used for initiation of tissue culture and the remaining part was snap frozen in liquid nitrogen and stored at -80°C for subsequent molecular cytogenetic analysis.
Figure 1 Clinical information of 37-year-old female patient. A, Brain enhanced CT scan demonstrated an irregularly enhanced tumor node with cyst in right temporal lobe. B showed 1st recurrence tumor relapsed and invaded posteriorly, superiorly to parietal lobe. (more ...)
Human glioma cell line SHG-44 (P53 mut) was derived from a poorly differentiated astrocytoma of the left frontal lobe of a 32-year-old woman who was undergoing surgical removal of a right temporal tumor at the Department of Neurosurgery, the 2nd Affiliated Hospital of Soochow University School of Medical (Suzhou, China). It was the first established human glioma cell line in China according to the library and information system of the Chinese Academy of Sciences, and has been widely used in human glioma research in China [1
]. Human glioma cell line U251 (P53 mut) was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Phoenix cells were the gift from Dr. Gu JM (Vanderbilt University, Nashville, Tennessee). All cell lines were cultured in Dulbecco's Modified Eagle Medium (Invitrogen, California, U.S.) supplemented by 10% heat-inactivated fetal calf serum (Si Ji Qing, Hangzhou, China).
Culture of Brain tumor stem cell spheres, neural stem cell spheres and multicellular tumor spheres
Human glioma tissues were obtained from a 52-year-old female patient who underwent two operations within 2 months for rapid relapse of human malignant gliomas. Both primary and recurrent lesions were pathologically diagnosed as mixed glioma consisting of anaplastic ependymoma and astrocytoma. Human fetal brain tissue was obtained from consent-informed women who received induced abortion in accordance with the protocol approved by Ethical guidelines for the use of human embryonic or fetal tissue for experimental and clinical neurotransplantation and research of Network of European CNS Transplantation and Restoration (NECTAR) [4
The fresh tumor specimens was washed, deprived of vessels, acutely dissociated in oxygenated artificial cerebrospinal fluid and subjected to enzymatic dissociation as described previously [5
]. Tumor cells were then re-suspended in serum-free DMEM/F-12 containing human recombinant N2, EGF and bFGF (20 ng/ml; Invitrogen, California, U.S.), and plated at a density of 3 × 106 live cells/60-mm plate. Cells were fed by changing half of the medium every 3 days. Brain tissue from a 1-month-old abortive dead embryo was subjected to the same procedure as described above to get neural stem cell spheres [6
]. SHG44 and U251 cells were planted in liquid media over an agar base to form multicellular tumor spheres [7
Construction of tissue microarray and Immunohistochemical analysis
A total of 71 gliomas of the cerebral hemisphere, resected at the 1st and 2nd affiliated Hospital of Soochow University were studied. These tumors were from 40 men and 31 women, ranging in age from 4 to 80 (mean, 41.04) years at diagnosis. All of these patients did not receive chemotherapy and radiation before surgical removal. Tumors were classified and graded using the World Health Organization (WHO) scheme (2000) (grades I–IV) [8
]. Eleven normal brain tissues were gathered from consent-informed individuals who underwent partial removal of the temporal lobe for cerebral decompression. SHG44 multicellular tumor spheres (n = 4), GBM multicellular tumor spheres (n = 4), brain tumor stem cell spheres (n = 4) and neural stem cell spheres (n = 4) were centrifuged and embedded in melted 3% agarose gel to form agarose cell blocks (ACBs) [9
]. Tissue microarray (TMA) technique was used for immunohistochemical study [10
]. The tumor samples and cell blocks were fixed in 4% phosphate buffered formaldehyde and processed into paraffin blocks with standard methods. Histologically representative tumor regions of hematoxylin-eosin (HE) stained slides were selected by a neuropathologist (Professor Liu zhen yan) and corresponding areas were sampled in tissue microarray blocks with the aid of manual tissue microarrayer (Bo Nan Biotechnology Inc. Shanghai, China) according to the manufacturer's instruction. The sample diameter of the tissue core in the microarray block was 600 μm.
Tissue microarray sections were washed in PBS and incubated for 1 h with rabbit anti CDC2 polyclonal antibody (Santa Cruz Biotechnology), rabbit anti CyclinB1 polyclonal antibody (Santa Cruz Biotechnology) or with 1% BSA-PBS as a negative control. After washing, the slides were incubated with biotinylated anti-rabitt immunoglobulin (LSAB) for 30 min, washed again, and incubated with horseradish peroxidase-conjugated streptavidin for 30 min. The reaction was revealed by 3, 3'-diaminobenzidine and counterstained with hematoxylin.
Evaluation of slides
Antigen expression was defined as the presence of nuclear staining on tumor cells for CDC2, CyclinB1 and TUNEL. An immunoreactive score (IRS) was applied. The IRS (negative (-), 0~2; weak positive (+), 3~4; positive (++), 5~6; strong positive (+++), >7) was the product of staining intensity (graded between 0 and 3) and the percentage of positive cells (graded between 0 and 4: 0, ≤5%; 1, 6%~25%; 2, 26%~50%; 3, 51%~75%; 4, ≥75%) [11
]. Five vital tumor fields were evaluated (magnifications 400×) and a final mean score for each tumor was achieved.
Construction of recombinant retroviral vectors expressing short hairpin RNAs and transfection in vitro
The pSUPER.retro.puro purchased from OligoEngine was a vector system for expression of short interfering RNA. Recombinant retroviral vectors expressing short hairpin RNAs were constructed according to the product manual. Three pairs of oligonucleotides were designed. The shRNA1, 3 corresponds to positions 256–274, 789–807 of the CDC2 open reading frame, respectively. The oligoengine workstation software gives the effect target corresponds to positions 908 of the CDC2 open reading frame. But we designed C4 target corresponds to positions 909 (909–927). We want to know whether it was an "inactive target" (Figure ). These forward and reverse oligos were annealed and cloned into the vector between the unique BglII and HindIII enzyme sites. The presence of the correct insert of recombinant pSUPER.retro vectors (C1, C3, C4) were confirmed by sequencing (BIO BASIC Inc, Shanghai, China; Sequencing primer 5'-GGA AGC CTT GGC TTT TG-3').
Figure 2 Schematic depiction of three shRNA: Three pairs of oligonucleotides were designed and were synthesized by BIO BASIC Inc (Shang Hai, China). shRNA1 corresponds to positions 256–274 of the CDC2 open reading frame, shRNA3 to positions 789–807 (more ...)
Glioma cell lines were transfected with recombinant retroviral vectors using a modified Lipofectamine™ 2000 protocol (Invitrogen, California, U.S.), in which the quantity of plasmid DNA was double of the recommended quantity in order to achieve the highest transfection efficiency. Cells were harvested 48 h after recombinant retroviral vectors transfection for RT-PCR, Western blots, cell cycle analysis and apoptosis analysis.
Cell proliferation assay in vitro
SHG44 and U251 cells were plated in 24-well plates (Corning, NY, U.S.A) at 1.5 × 105 per well. Twenty four hours after transfection, cells were transferred into a 50 ml cell culture flask (Corning). Time kinetics of cell proliferation was conducted at 24, 48, 72 and 96 h after transfection with recombinant retroviral vectors. Cells were harvested with 0.25% trypsin, 1 mM EDTA in phosphate buffered saline (PBS) and stained with Trypan blue. The number of cells was counted with a microscope using a haemacytometer.
RT-PCR and real time PCR
Total RNA was isolated from cells using TRIzol reagent (Gibco, California, U.S.A) according to the manufacturer's instructions. Total RNA (5 μg) was reverse transcribed using 1 μg oligodT primer with MMLV reverse-transcriptase (Promega) in a 40 μ1 reaction volume containing 1.25 mM deoxyribonucleoside triphosphate (dNTP) at 42°C. PCR was performed using 1 μl of cDNA in 50 μl PCR reaction buffer containing 15 pmol of each primer, 0.1 mM dNTP, and 0.3 units Taq polymerase (Promega). Primer sequences used and conditions of these reactions were as follows: CDC2: sense 5'-GGT TCC TAG TAC TGC AAT TCG-3'; antisense 5'-TTT GCC AGA AAT TCG TTT GG-3', (94°C 1 min, 51°C 1 min, 72°C 1 min, ×30 cycles, 709 bp as PCR product), Cyclinb1: sense5'-CAG TCA GAC CAA AAT ACC TAC TGG GT-3'; antisense 5'-ACA CCA ACC AGC TGC AGC ATC TTC TT-3', (94°C 30 s, 54°C 30 s, 72°C 30 s ×30 cycles, 191 bp as PCR product), β-actin (internal control): sense 5'-TCC TGT GGC ATC CAC GAA ACT-3'; antisense 5'-GAA GCA TTT GCG GTG GAC GAT-3'(314 bp as PCR product). The products were electrophoresed through 2% agarose gel.
cDNA was used to perform real-time quantitative PCR using MJ Research PTC-100 with TaKaRa Ex Taq R-PCR Version 2.1 (TaKaRa, Da lian, China) and EvagreenTM (Biotium, Inc., CA, U.S.A) according to product's instruction. Primer sequences are shown as follows: CDC2: sense 5'-CAG TCT TCA GGA TGT GCT TAT GC-3'; antisense 5'-GAG GTT TTA AGT CTCTGT GAA GAA CTC-3'. β-actin: sense 5'-AGC GAG CAT CCC CCA AAG TT-3'; antisense 5'-GGG CAC GAA GGC TCA TCA TT-3'.
Protein extraction and western blot analysis
Cells were harvested using trypsin:EDTA and lysed in TRIzol reagent (Gibco). Proteins were precipitated from the phenol-ethanol supernatant with isopropyl alcohol obtained after precipitation of DNA with ethanol and dissolved in 1% SDS by pipetting. Protein concentrations in solutions were determined using the Bradford protein assay. PageRuler™ Prestained Protein Ladder (5 μl) (Fermentas Inc. MD, U.S.A) and protein solutions containing 40 μg per lane were electrophoresed using SDS-PAGE (15% polyacrylamide gel) and blotted onto nitrocellulose (NC) membrane. Membranes were blocked with 5% fat-free milk solution. Samples were probed with 1:200 dilution of a Rabbit polyclonal antibodies against CDC2 (Santa-Cruz Biotechnology, Inc., Santa Cruz) and a 1:5000 dilution of goat anti-rabbit HRP-conjugated IgG (Hua mei Chemical Corp., China). Rabbit monoclonal antibody against GAPDH (Affinity BioReagents, Inc.) was used as a loading control. Bands were revealed using ECL kit (Pu fei Chemical Corp., Shang hai, China).
Analysis of cell cycle and apoptosis
Cell viability was assessed by Trypan blue staining. Cell cycle was analysed using a FACScan flow cytometer (Becton Dickinson). Briefly, cells were washed with PBS, treated with RNase A, and stained with PI (Sigma). Apoptosis was assessed using rh Annexin V FITC Kit according to manufacturer's instructions (Bender Medsystems). The data were acquired using Cell Quest V.3.3 software (BD). To detect apoptotic cells in xenogeneic graft tumors, sections were analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) (Roche Molecular Biochemicals), according to the manufacturer's instructions.
The effective recombinant retroviral vectors were packaged in Pheonix cells by standard calcium phosphate transfection protocol [12
]. Virus supernatant was collected 48 hours post-transfection, filtered to remove nonadherent cells and cellular debris, frozen in small aliquots on dry ice, and stored at -80 C. Detection of virus titer was refer to the methods of Short Protocols in Molecular Biology [13
Mouse Models of Gliomas and Recombinant Retrovirus Injections
Nude mice with nu/nu gene from the NC strain (Fujita Health University, Japan) were bred and maintained in our Specific Pathogen Free Animal Care Facility. Female mice (6- to 8-week-old) were used in this study. Subcutaneous injections of 5 × 106 tumor cells in 0.2 ml solution were made into the flanks of recipient nude mice, as described previously [14
]. Eight mice were included in control and experimental groups. When the diameter of xenogeneic graft tumors was about ~0.5 cm, recombinant retrovirus was directly injected into xenogeneic graft tumors by using the following parameters: 10 μl per site, 10 μl infusion rate/min, 100 μl per one injection, injection per 3 days and total 3 times (total 300 μl). Twenty days after the last injection, the difference of tumor weights between control group and experimental group was compared.
To determine if CDC2-depletion prolonged animal survival, 1 mm3 SHG44 xenogeneic graft tumor tissue was injected into the frontal lobe of 6-week-old nude mice by using the following parameters: 0.5-mm anterior, 2.5-mm lateral, 2.5-mm depth. After 3 days, animals were treated with a single intratumoral injection of PBS and recombinant C3 retrovirus (n = 8/group 20 μl per mice, 0.5 μl/min infusion rate).
The relationship between expression rates of CDC2 and pathologic grades of human gliomas was analyzed with correlation coefficient. The Spearman's Rank Correlation was used to measure the correlation between CDC2 and CyclinB1 expression in gliomas. Statistical analyses ex vivo were performed using the Student's t test. Statistical analyses in vivo were performed using the Wilcoxon signed-rank test. All the reported P-values are 2-sided and a value of P < 0.05 was considered statistically significant.
Statement of ethics
Research that is reported in the manuscript has been performed with the approval of ethics committee of 2nd affiliated hospital of Soochow University. Animal studies were approved by the Soochow University of Animal Care and Use Committee and followed internationally recognized guidelines.