The human cancer (Jakurt, H1299, A549, A427, H441, H1703, H322, H460, HCT116, H1975, H322, H358, H838, H28, H2052, Hela and H1650) and normal lung (WI-38 and CCL-211) cell lines were obtained from American Type Culture Collections (Manassas, VA). H290 and MS-1 cell lines were obtained from NIH (Frederick, MD). Cells were grown in complete growth medium (Dulbecco's modified Eagle's medium for HeLa, A549 and CCL-211; Eagle's Minimum Essential Medium for WI-38 ; Roswell Park Memorial Institute's medium for H1299, A549, A427, H441, H1703, H322, H460, HCT116, H1975, H322, H358, H838, H28, H2052 and H1650) supplemented with 10% fetal bovine serum, 10 units/ml penicillin and 10 µg/ml streptomycin at 37°C and 5% CO2.
Fresh tumor tissues and adjacent normal tissues were obtained from patients with non-small cell lung cancer (NSLC) who were undergoing surgical resection of the primary tumor. The study was approved by the University of California, San Francisco, institutional review board (CHR# H8714-11647-14). We obtained written informed consents from all participants involved in our study. Tissue samples were kept at −180°C liquid nitrogen freezers before use, and final pathologic diagnosis was confirmed by a pathologist at the University of California, San Francisco (UCSF), USA. Normal adult genomic DNA form peripheral blood was purchased from BioChain (Hayward, CA).
Fluorescence-in situ hybridization
The fluorescence-in situ hybridization (FISH) probe for CSNK2A1P (RP11-567I13, chromosome 11p15.3) was purchased from BACPAC Resources (Oakland, CA). The chromosome 11 centromere was labeled by Vysis CEP 11 SpectrumGreen™ probe (Abbott Molecular, Abbott Park, IL). Metaphase slides were prepared using standard protocols of the UCSF Molecular Pathology Core facility. All hybridizations were done by the UCSF Molecular Pathology Core facility. The CSNK2A1P BAC probe was labeled with Cy3 red by nick translation. Probe mixture was prepared according to the standard protocol.
DNA and cDNA sequencing analysis
Genomic DNA or total RNA was isolated from cell lines and tissue samples using the DNeasy Blood & Tissue Kit or the RNeasy Mini Kit (Qiagen Valencia,CA), respectively. The CSNK2A1P gene was PCR amplified using its gene-specific primers. The forward and reverse primers used for PCR and sequencing were: 5′-AGAAAATTGCTCCCCACTCC-3′ and 5′-GTGCTGCCAGAGAATGA CAA-3′ respectively. The PCR products were gel-purified using the QIAquick Gel Extraction kit (Qiagen Valencia,CA) and were subsequently sequenced at MCLab (South San Francisco, CA).
Semi-quantitative reverse transcription-PCR (RT-PCR) analysis
Total RNA from cell lines and tissues was isolated using an extraction kit, and DNA was eliminated by on-column treatment with DNase (RNeasy Mini kit; Qiagen, Valencia, CA, USA). Semiquantitative RT-PCR was performed by using SuperScript One-step RT-PCR with Platinum Taq kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. One-step RT-PCR was performed using pairs of CSNK2A1P-specific primers (Forward: 5′-AGAAAATTGCTCC CCACTCC-3′ and Reverse: 5′-GTGCTGCCAGAGA ATGACAA-3′), CSNK2A1-specific primers (Forward: 5′-TGGGGACAGAAGATTTATATGA-3′ and Reverse: 5′-CTGAAGAAATCCCTGACA TCAT-3′) and CSNK2B-specific primers (Forward: 5′-CAGGTCCCTCACTACCGACA -3′ and Reverse: 5′-CAGCTGGTAGGCCATCGGAT-3′). The PCR products were verified by direct DNA sequencing. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as an internal control. Amplification conditions for CSNK2A1P, CSNK2A1 and CSNK2B were as follows: 1 cycle of 45°C for 30 min, followed by 1 cycle of 95°C for 5 min, and 35 cycles of 95°C for 1 min, 56°C for 1 min, 72°C for 1 min and 1 cycle of 72°C for 10 min then 4°C.
Cloning of CSNK2A1P cDNAs
The full-length cDNA of CSNK2A1P gene from A549 cells was cloned using TOPO TA Cloning Kit (Invitrogen Carlsbad, CA), and then was subcloned into the HindIII and BamHI sites of the pcDNA3.1/myc-His vector (Invitrogen, Carlsbad, CA). The forward and reverse primers used for cloning were 5′-CCTTAAAAGCTTGACCATGTCGG GACCCGTGCCAAG-3′ and 5′-CCTTAAGGA TCCGACTGCTGAGCGCCAGCGGCAG-3′′respectively. The CSNK2 A1pcDNA3.1/myc-His vector was a gift from Dr. L. A. Pinna. Both myc-His tagged CSNK2A1 and CSNK2A1P genes were subsequently cloned into the SnaBI site of the pBabe-puro vector. The forward and reverse primers used for cloning were 5′-GGCTAGTTTACGTAG ACCATG TCGGGACCCGTG CCAAG-3′ and 5′-AAGGCACAGT CGACGCT GATCAGCGGGTT TAAACTCA-3′ respectively. All resultant vectors were verified by direct DNA sequencing.
Retroviral production and transduction
The CSNK2A1 and CSNK2A1P retroviral vectors were then transfected into the HEK 293 Phoenix ampho packaging cells (ATCC, Manassas, VA) by using Fu-GENE6 transfection reagent (Roche, Lewes, UK) to produce retroviral supernatants. Forty-eight hours after transfection, the supernatant was filtered through a 0.45 µm syringe filter. Retroviral infection was performed by adding filtered supernatant to mesothelioma cell lines cultured on 10cm dishes with 50% confluent in the presence 8 ug/ml of polybrene (Sigma, St. Louis, MO). Six hours after infection, the culture medium was replaced with fresh medium and infected cells were allowed to recover for 48 hours. Infected cells were selected by adding 1µg/ml puromycin (Sigma, St. Louis, MO) to the culture medium for 48 hours and then maintained in complete medium with 0.5µg/ml puromycin. Empty retroviral infected stable cell lines were also produced by the above protocols.
Western blot analysis
Whole protein was extracted by M-PER Mammalian Protein Extraction Reagent from cell lines added with Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA) and Complete Protease Inhibitor Cocktails (Roche, Lewes, UK) according to manufactures' protocols. The proteins were separated on 4–15% gradient SDS–polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bellerica, MA). The following primary antibodies were used: anti-CK2α, anti-β-actin (Sigma Chemical, St. Louis, MO), anti-PML (Santa Cruz, Santa Cruz, CA), and anti-Myc tag (Cell Signaling, Danvers, MA). After being incubated with appropriate secondary antibodies, the antigen-antibody complexes were detected by using an ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ).
Colony formation assay
NIH3T3 cells stably transfected with CSNK2A1 or CSNK2A1P (5×102) were plated in 10 cm culture dishes and incubated in complete medium for 14 days. The colonies were then stained with 0.1% crystal violet, and colonies greater then 50 cells were counted. Results were expressed as relative colony formation: percentage of the number of colonies relative to the empty vector transfected controls. Three independent experiments were performed.
Soft agar growth assay
For experiments using the pcDNA3.1/myc-His vectors expressing CSNK2A1 and CSNK2A1P, NIH3T3 cells were transfected with these vectors or control empty vector and then selected in 100 ug/ml of G418 for 1 week. Cells (1×103) were then cultured in DMEM plus 15% FBS in 0.35% (w/v) low melting temperature agar between layers of 0.7% low melting temperature agar. After 4 weeks, colonies were stained with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co.), and colonies containing >100 cells were scored. Colonies were photographed and counted after staining.
Protein degradation assay
The stable NIH3T3 cells retrovirally transfected with CSNK2A1 and CSNK2A1P were plated on 6 cm culture dishes. At 80% confluence, cells were exposed to 20 µg/ml cycloheximide and harvested at different time points (0, 2 and 6 hours). Total cellular proteins were extracted and were analyzed by western blot analysis using β-actin as the loading control.
293T cells were transiently co-transfected with PML pcDNA4/V5-His (a gift from Dr. Zheng Pan) and CSNK2A1 or CSNK2A1P pcDNA3.1/myc-His vectors with Lipofectamine 2000 transfection reagent (Invitrogen Carlsbad, CA). Twenty-four hours after transfection, cells were treated with 10µM of MG132 (Sigma, St. Louis, MO) and then harvested in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 1% NP40), protease inhibitor, and phosphatase inhibitor cocktail (Roche, Lewes, UK). Immunoprecipitation was performed by the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore, Bellerica, MA) according to the manufacturer's protocols. Anti-Myc tag (Santa Cruz, Santa Cruz, CA) and anti-V5 tag (Invitrogen, Carlsbad, CA) antibodies were used for immunoprecipitation respectively.
Transfection of small interfering RNA
Pre-designed and validated CSNK2A1P and universal negative control small interfering RNAs (siRNA) were purchased from Invitrogen (Carlsbad, CA). Transfection was performed using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer's manual. Cells were plated in 60-mm dishes in antibiotic-free media and transfection was performed with cells at 60% confluence with a final concentration of 50 nM for each siRNA. At 72 hours after transfection, cells were analyzed for gene and protein expression.
Allele-specific amplification assays
The allele-specific amplification was measured using the ABI PRISM 7700 sequence detection system. PCR reactions for allele-specific expression (5 µl) contained 10 ng genomic DNA, 1× TaqMan universal PCR master mix, forward and reverse primers (900 nM), 200 nM VIC-labeled probe and 200 nM FAM-labeled probe. Amplification conditions were as follows: 1 cycle of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 63°C for 1 min. The data was analyzed using the Allelic Discrimination Sequence Detection Software (Applied Biosystems). The TaqMan primers and probes were custom designed using the Primer Express Oligo Design Software (Applied Biosystems). Probes were MGB probes were designed specifically for TaqMan Allelic Discrimination (Applied Biosystems). Primer sequences are 5′-CCGCCTTGGTTTTTGAACAC-3′and 5′-GGCCTTCAGAATCTCATACATGTAAA-3′; probe sequences are FAM-CACAGACTATGACTC (398C allele specific) and VIC-TCACAGACTATGATTC (398T allele specific). PCR was done in triplicate for each sample and experiments were repeated at least three times. CT values were normalized to the average normal genomic CT difference in each experiment. The CT value differences between the two probes for the triplicates were then averaged.
For determination of the kinase activity of the expressed CSNK2A1 and CSNK2A1P proteins, Casein Kinase 2 Assay Kit (Millipore, Bedford, MA) was used according to manufacture's protocol. Stable NIH3T3 cells transfected with the CSNK2A1 and CSNK2A1P genes were harvested in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 1% NP40), protease inhibitor, and phosphatase inhibitor cocktail (Roche, Lewes, UK). Immunoprecipitation was performed by the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore, Bellerica, MA) according to the manufacturer's protocols. Anti-Myc tag (Santa Cruz, Santa Cruz, CA) antibody was used for immunoprecipitation. Kinase assay was carried in a final volume of 50 µl containing 20 mM MOPS (pH 7.2), 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 15 mM Mgcl2, 200 µM for CK2 substrate peptide: RRRDDDSDDD (Millipore, Bedford, MA), and [γ-33P]-ATP. After incubation in 30°C for 20 minutes, assay was stopped by adding of 20 µl 4% trichloroacetic acid and transferred 25 µl to P81 phosphocellulose squares. After washing with 0.75% phosphoric acid for 6 times and with acetone for 1 time, phosphocellulose squares were dried and transferred to scintillation vials for counting.
The data are shown as mean values ± standard deviation (SD). Student's t-test was used to compare results between control and experimental groups in the colony formation assay. Chi-square test was used to compare the frequency of the CSNK2A1P polymorphisms between lung cancer tissues and normal controls. Pearson correlation coefficient was used to access the correlation between mRNA expression and copy numbers of the CSNK2A1P gene in cancer cell lines. Statistical analysis was carried out using SPSS (version 10.0, Chicago, IL). A P value of less than 0.05 was considered statistically significant. All statistical tests were two-sided.