Normal/Cancer cell cultures
Clones of colorectal cancer cells (HCT116Clone2_XRR, HCT116Clone10, and HCT116CloneK_XRS), normal fibroblast cells (COS-7 and AG1522), breast cancer cells (MCF-7 and MDA MB435), glioma cells (U251 MG), melanoma cells (SKMEL-3 and HT144), lung cancer cells (HTB56 and A549), ovarian cancer cells (A2780CP and A2780S), prostate cancer cells (PC-3 and DU145), leukemia cells (K-562), colorectal cancer cells (HT29), renal cancer cells (SN12C), and mouse fibrosarcoma cells (TM28) were grown in DMEM/Ham' s F12 1:1 mix (Wisent Inc., St. Bruno, QC, Canada) supplemented with 10% fetal bovine serum (FBS) (Wisent Inc.) and 15 mM HEPES in a humidified atmosphere of 95% air, 5% CO2 at 37°C. Various individuals from the ORCC, Ottawa, Canada, kindly provided us with the cells. They were: J.C. Bell (COS-7, PC-3, DU145, K-562, HT29, and SN12C cells), C.L. Addison (MCF-7 and MDA MB435 cells, neuroblastoma cells (Daoy, SKNA, and SHSY), normal human dermal microvascular cells (HDMEC)), HC Birnboim (TM28 cells), B. Vanderhyden (ovarian cancer cells OCCL, OV2008, C13, SKOV3, 433, HEY, and 429), and M. McBurney (mouse embryonic stem cells R1). H.C. Birnboim also donated a C57BL/6 mouse with a solid fibrosarcoma (TM28) growing in the flank. A. Gatignon (Lady Davis Insitute, Montreal, Canada) kindly provided the U251 MG cells. Clones of HCT116, AG1522, SKMEL-3, HT144, A2780CP, and A2780S cells were from our own laboratory collection (C.E. Ng, ORCC, Ottawa, Canada).
Cloning and sequencing of Hs XRRA1 splice variant
A specific primer pair was created to amplify the 5' region of the XRRA1 cDNA. They were 5'-GCGCTGGAGACACTGATGCTGG ATGACAAC-3' and 5'-GGCCAGGCTAAGGTATCTCAGCTCTGGG-3'. These primers generated a product of 452-bp that comprised the first 4 exons. Total RNA from HCT116Clone2_XRR and HCT116Clone10 cells were used as template for RT-PCR. PCR fragments were gel purified using Gel Extraction Kit (Qiagen Inc., Mississauga, ON, Canada) and cloned into pTZ57R plasmid using InsT/Aclone PCR Product Cloning Kit (MBI Fermentas, Burlington, ON, Canada). Sequencing of the clones was performed by DNA Sequencer model 4000L according to the manufacturer' s protocol (LI-COR Biosciences, Lincoln, NE, USA) using M13 primer.
Construction of macaque cDNA libraries and DNA sequencing of their cDNA inserts
Oligo-capped cDNA libraries were made from macaque brain and testis. cDNAs were isolated according to the method described previously [22, 23]. The 5'-end sequences of the clones were sequenced using ABI 3700 sequencer (Applied Biosystems, Tokyo, Japan) and categorized using DYNACLUST (Dynacom Co., Chiba, Japan) based on a BLAST search against the GenBank database. The entire sequences of clones were determined by the primer walking method with an ABI PRISM BigDye Terminator Sequencing kit (Applied Biosystems) according to the manufacturer' s instructions.
Construction of plasmid expressing Mf XRRA1 fused to GFP
Macaque cDNA clone of QtsA-20344 was found from the cDNA libraries to contain Mf XRRA1 transcript. A 1709-bp Xho I fragment of Mf XRRA1 was removed from pME18S-FL3 and sub-cloned into Xho I sites of pEGFP-C1 (BD Biosciences, Mississauga, ON, Canada), immediately downstream of the GFP sequence (pFM1709). To create a truncated Mf GFP-XRRA1 (pFM584), 1.1-kb Eco RI fragment was removed from pFM1709 leaving a 584-bp of the 5' XRRA1 cDNA fused with GFP.
Transfection of GFP-XRRA1 into HCT116Clone2_XRR, HCT116Clone10, HCT116CloneK_XRS, and COS-7 cells
HCT116 clones and COS-7 cell were transfected with a mixture of pFM584, pFM1709 or pEGFP-C1 and lipid complexes FuGENE 6 (Roche Diagnostics, Laval, QC, Canada) transiently or stably. Briefly, HCT116 clones and COS-7 were grown on cover slips in a 6-well plate and reached 60–70% confluences before transfection. The ratio of FuGENE 6 and pure plasmid was 6:1 in serum free medium. G418 (Sigma-Aldrich, Oakville Ltd., ON, Canada) at a concentration of 400 μg/ml was used for selection. Cells were fixed with 3.7% formaldehyde in PBS for 10 min, and then washed three times with 1x PBS. Coverslips were mounted upside down onto slides with Dako Fluorescent Mounting Medium (DAKO, Carpinteria, CA, USA). Visualization was performed using a fluorescence microscope Axioskop 2 MOT (Carl Zeiss MicroImaging Inc., Thornwood, NY, USA). Images of cells were captured with a CCD camera fitted with a FITC filter.
Immunocytochemistry
Transfected COS-7 and HCT116Clone2_XRR cells with pFM1709 or pEGFP-C1 were grown on cover slips until they reached 70–80% confluency before being treated with 4 or 10 Gy XR. Cells were fixed in 2% formaldehyde in 1x PBS for 15 min 24 h after XR treatment. Cells were then permeabilized with 0.2% Triton X-100 for 10 min. Ku86 antibody was obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. The secondary antibody was conjugated with Cy3 (Amersham Biosciences Corp, Baie d' Urfe, QC, Canada). Controls for the immunostaining were the secondary antibody alone, and control for the experiment was α-Tubulin antibody that was obtained from Calbiochem, San Diego, CA, USA.
RT-PCR
Total RNAs of normal/cancer cells were extracted using RNAeasy kit (Qiagen Inc.) according to the manufacturer' s instructions. TRIZOL Reagent (Invitrogen Inc., Burlington, ON, Canada) was used to isolate RNA of mouse fibrosarcoma tumor biopsy. RNAs of human peripheral blood leukocyte, spleen, thymus, small intestine, colon, ovary, testis and prostate were purchased from BD Biosciences. First Strand cDNA Synthesis kit (MBI Fermentas) was used to create first strand cDNA from RNA samples. A specific primer pair that amplified ~1 kb house keeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as the control. The primers were 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATGTAGGCCA TGAGGTCCACCAC-3'. Conditions of PCR were as follows: 94°C for 30 s, 61°C for 15 s, and 72°C for 40–60 s for 35 cycles. The PCR products were then separated on 1.5% agarose gels, stained with ethidium bromide, and visualized with the gel documentation imager Epi Chemi II (UVP Inc., Upland, CA, USA). Densitometry of bands were analyzed using Labworks™ software (UVP). Relative expression of XRRA1 was calculated as percentages of the mean raw densities of grayscale level of XRRA1 to G3PDH.
X-radiation of HCT116Clone2_XRR, HCT116Clone10, and HCT116CloneK_XRS cells
Clones of HCT116 cells were cultured at 1 × 105 cells/ml. Cultures reached 70–80% confluence before being treated with XR. Each clone was treated with a single dose of 4 or 10 Gy using a 250 kVp X-ray unit (Pantak, CT, USA) at a dose rate of 150 cGy/min. Total RNAs were collected 5 minutes, 6 hours and 24 hours following the XR. Specific primers for Hs XRRA1 as mentioned above were used. G3PDH was used as control. Relative expression of XRRA1 to G3PDH was evaluated
cDNA microarray of Mf XRRA1 from testis versus mixed tissues or human testis
512 fully sequenced testis cDNA clones were amplified using 5'-CTTCTGCTCTAAAAGCTGCG-'3 as a forward primer and 5'-CGACCTGCAGCTCGAGCACA-'3 as a reverse primer. Successful amplification was confirmed by agarose gel electrophoresis. Approximately 300 μg /ml DNA in 2 × Solution-T reagent (Takara Bio Inc., Shiga, Japan) were printed on duplicate glass-slides with a GMS 417 arrayer (Genetic MicroSystems, Woburn, MA, USA). RNA was isolated with TRIZOL Reagent (Invitrogen K.K., Tokyo, Japan) and purified with Oligo-Tex (Takara Bio Inc.). Macaque testis RNA was labeled with Cy3-dUTP (Pharmacia K.K., Tokyo, Japan). A mixture of macaque RNA was also obtained from 10 tissues: brain, heart, skin, liver, spleen, renal, pancreas, stomach, small intestine, large intestine, and labeled with Cy5-dUTP (Pharmacia). We mixed equal amount of RNA of these ten tissues. In a separate experiment, RNA from human testis (BD Biosciences) was labeled with Cy5-dUTP. Both labeled RNAs were co-hybridized to DNA spots. After the hybridization and washing procedure, slides were scanned with ScanArray (PerkinElmer Life Sciences Co., Tokyo, Japan). Beta actin and/or total signal intensity was used as the control for hybridization. The methods and results of DNA microarray have been deposited in the Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo with the accession numbers GPL206, GSM2387, and GSM2388.
ESTs-based ORF assembling (EBOA) and other in silico analysis
In order to elucidate what gene R40588 might represent, we employed EBOA to obtain the full-length cDNA containing start-, stop-codon, and poly-A signal or a complete open reading frame (ORF). The sequence of R40588 was used as a template to do BLAST search for all possible overlapping ESTs from human dbEST http://www.ncbi.nlm.nih.gov/dbEST. These retrieved ESTs, either sense-antisense or 5'- and 3'-direction, were selected and were again used as template for searching other overlapping ESTs. To confirm the ORF candidates, (i) we carried out a BLAST search against full-length cDNAs that were available on public databases, and (ii) genomic sequences comprising the ORF candidates were analyzed with GrailEXP version 3.3 http://compbio.ornl.gov/grailexp[11]. Multiple alignments were done according to ClustalW method [24]. Transcription factors and their binding sites were predicted by employing the Alibaba2 version 2.1 program http://www.gene-regulation.com/pub/programs.html that used the TRANSFAC database http://transfac.gbf.de/TRANSFAC[25]. Possible tissues/organs expression of XRRA1 was explored from the SAGEmap http://www.ncbi.nlm.nih.gov/SAGE[26]. Protein motifs were searched using software available on these web sites: http://www.expasy.ch/prosite[27], http://www.at.embnet.org/embnet/tools/bio/PESTfind[28], and http://www.cbs.dtu.dk/services/NetPhos[29].