LN229 (glioblastoma, ATCC#CRL-2611), U87MG (glioblastoma/astrocytoma, ATCC#HTB-14), Hela (cervix ADCA, ATCC#CCL-2), Caco2 (colorectal ADCA, ATCC#HTB-37), DLD1 (colorectal ADCA, ATCC#CCL-221), HCT116 (colorectal ADCA, ATCC#CCL-247), SW480 (colorectal ADCA, ATCC#CCL-228), A549 (lung ADCA, ATCC#CCL-185), LNCaP (prostate ADCA, ATCC#CRL-1740), MCF7 (breast ADCA, ATCC#HTB-22), A431 (squamous cell carcinoma, ATCC#CRL-1555), NCI-H69 (SCLC, ATCC#HTB-119), NCI-H82 (SCLC, ATCC#HTB-175), NCI-H345 (SCLC, ATCC#HTB-180), NCI-H446 (SCLC, ATCC#HTB-171), NCI-H510A (SCLC, ATCC#HTB-184), NCI-H1436 (SCLC, ATCC#CRL-5871), and NCI-H1930 (SCLC, ATCC#CRL-5906) were originally purchased from ATCC and stocked in our Research Center. TE1 (esophagus squamous cell carcinoma), TE3 (esophagus squamous cell carcinoma), TE4 (esophagus squamous cell carcinoma), TE5 (esophagus squamous cell carcinoma), and TE10 (esophagus squamous cell carcinoma) were gifts from Dr. Sasaki (National Cancer Center Research Institute). SBC-3 (SCLC, #JCRB0818) was obtained from the JCRB and stocked in our Research Center. All cell lines were cultured in cell culture dishes (BD Biosciences) at 37°C and 5% carbon dioxide using RPMI 1640 (SIGMA), DMEM (SIGMA) supplemented with 10% fetal bovine serum (FBS, Nichirei Bioscience), or HITES Medium [25, 26] supplemented with penicillin/streptomycin (Invitrogen). For the PTN assay, 100 ng/ml of recombinant human pleiotrophin/PTN (R&D Systems #252-PL) was used.
Human cancer samples
Samples were obtained with informed consent from each individual, and the study was approved by the Ethics Committee of the National Cancer Center East Hospital. During the period from January 1992 to December 2010, a total of 252 patients who had primary tumors were treated at the National Cancer Center Hospital East, Chiba, Japan. All primary cancers with a pathologic diagnosis based on the classification schema of the WHO classification were reviewed, with 105 cases as adenocarcinoma (ADC), 61 as squamous cell carcinoma (SQCC) and 86 as neuroendocrine tumors (NETs). We used tissue microarray (TMA) to measure PTPRZ1 expression within lung tumors . Each case in which more than 80% of the cancer cells reacted positively for an antibody to PTPRZ1 was recorded as positive.
Antibodies used included anti-PTPRZ1 (SIGMA #015103) , anti-Phosphotyrosine, clone 4 G10 (Millipore #05-321), anti-Calmodulin (Santa Cruz sc-5537, Millipore #05-173, abcam ab45689), anti-phospho-Calmodulin (Santa Cruz Biotechnology sc-23760-R, Millipore #09-295) and anti-β-tublin (Cell Signaling #2146).
All immunohistochemical (IHC) analyses were performed on paraffin-embedded tissues obtained from the primary tumor in the surgical specimen. For all IHC analyses the surgically resected specimens were fixed in 10% formalin and embedded in paraffin for routine pathological examination. We prepared and used 5-μm-thick paraffin sections cut from a paraffin block containing histological findings that were representative of the tumor. The procedure for IHC was previously described [27, 28]. Antigen retrieval was performed in citrate buffer solution (pH 6.0). Endogenous peroxidase was blocked with 0.3% H2O2 in methanol for 15 min and all slides were heated to 95°C by exposure to microwave irradiation for 20 min and then cooled at room temperature (RT). Slides were washed in PBS and after a 1 h incubation at RT with the primary antibodies, the slides were incubated for 30 min with a labeled polymer EnVision TM+, Peroxidase–conjugated anti-Mouse or Rabbit (Dako, Tokyo, Japan). The chromogen used was 2% 3, 3′-diaminobenzidine (DAB) in 50 mM Tris-buffer (pH 7.6) containing 0.3% hydrogen.
RNA isolation and real-time RT–PCR
Cells were washed with PBS and total RNA from the cell lines was isolated with TRIzol Reagent (Invitrogen). Complementary DNA (cDNA) was synthesized using the PrimeScript® RT reagent Kit (TaKaRa, Japan). Real-time RT–PCR was carried out with specific primers and a Smart Cycler (Cepheid, Sunnyvale, CA, USA). Real-time fluorescence monitoring of the PCR products was performed with SYBR Green I fluorescent dye (TaKaRa). The levels of expression of specific genes are reported as ratios to the level of expression of GAPDH in the same master reaction. Synthesized primers were purchased from TaKaRa Bio with Primer Set ID given as PTPRZ1, 3′ (HA082543). GAPDH was used for normalization as control and the relative quantitation value compared to the calibrator for that target is expressed as 2-(Ct-Cc).
Western blotting was performed as described . After lentivirus infection with the vector for shLUC or shPTPRZ1, total cell lysate was prepared from cells cultured in complete medium. Primary antibodies were used at 1:1000 dilution and β-tubulin was used as loading control.
Expression of short hairpin RNA (shRNA)
Plasmid construction was carried out with Gateway system (Invitrogen) according to the manufacturer’s instructions. Cloning vectors were pDNOR221 (Invitrogen) and pENTR/U6 (Invitrogen). The lentiviruses were produced using 293FT cells (Invitrogen) transfected with pCAG-HIVgp, pCMV-VSV-G-RSV-Rev, and a lentivirus vector based on CSII-CMV-RfA-IRES2-Venus (Dr. Miyoshi, RIKEN BioResource Center) expressing shRNA with the sequence described below. Transfection was achieved using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Lentivirus-containing medium was filtered through a 0.45 μm filter and used for transduction of target cells. The sequences and plasmid names were; shLUC: GTGCGCTGCTGGTGCCAAC (pGL3, firefly luciferase), shPTPRZ1_1: GCCTATAAATTGTGAGAGCTT (pHMA017), shPTPRZ1_2: GCTGCTTTAGATCCATTCATA (pHMA019), and shPTPRZ1_3: GGATGGCAAACTGACTGAT (pHMA022).
Cells were incubated with anti-PTPRZ1 antibody (SIGMA) and excess antibody was removed by washing with PBS containing 2% FBS. Polyclonal goat anti-rabbit immunoglobulin conjugated to Phycoerythrin (PE) (Jackson) was added as a secondary antibody. The cells were then washed with PBS and flow cytometric analysis was performed using a FACSCalibur and FACSAria (BD Biosciences).
All of experimental SCID mice were handled in accordance with institutional guidelines established by the Animal Care Committee of the National Cancer Center East Hospital. H69 and H1930 SCLC cells expressing shRNA were injected into the subcutaneous tissue of SCID mice (7–8 weeks of age, CLEA, Tokyo, Japan). Tumor volume was calculated as the product of a scaling factor of 0.52 and the tumor length, width, and height were measured every week. For IHC analysis, organs were obtained from mice at 5 or 8 weeks after injection and fixed in 10% formalin.
Standard Student’s t-test was used to determine the significance between non-targeting control and shPTPRZ1 experiments. Statistical correlation was carried out using χ2test for independence (2 × 2 contingency table). P < 0.05 was considered statistically significant.