under-expression results in CDC25A overexpression in colon cancer [24]. Here we report the identification of a novel, alternatively spliced CDC25A isoform that resulted in the deletion of codon 110 termed CDC25AQ110del. We show that CDC25AQ110del is expressed at high levels in 75% of the NSCLC cell lines. CDC25AQ110del protein had higher stability and more nuclear distribution. Cells expressing high level of CDC25AQ110del were more resistant to UV irradiation. In patients with NSCLC, higher CDC25AQ110del levels in the tumors were associated with poor clinical outcome. Our data indicate that CDC25AQ110del expres- sion is common in NSCLC and may play a role in lung tumorigenesis and cancer progression. Materials and Methods Cell lines HEK293 and NSCLC cells were obtained from ATCC (Manassas, VA), and maintained in DMEM - 5% fetal bovine serum. Immortalized human bronchial epithelial cell lines (HBEC), HBEC2, HBEC3, HBEC4 and HBEC5 (gift from Drs. John Minna and Jerry Shay of the University of Texas Southwestern Medical Center, Dallas, Texas) [25], were main- tained in keratinocyte serum-free (KSF) media with recombinant human epidermal growth factor (rEGF) and bovine pituitary extract (Invitrogen, Carlsbad, CA). Plasmid transfection was performed using lipofectamine 2000 (Invitrogen). Western blotting Cells were harvested in RIPA buffer with protease inhibitor (Roche Bioscience), and separated by SDS-PAGE. Primary antibodies against CDC25A (clones 144 and F-6), cdc2 p34 (H- 297), Chk1, GAPDH (Santa Cruz Biotechnology, CA), phospho- Chk1(Ser345) (Cell Signaling Biotechnology, Danvers, MA), phospho-CDK1(Tyr15) (Calbiochem EMD chemicals Inc, Gibbs- town, NJ) were used. NE-PER protein extraction kit (Pierce Biotech, Rockford, IL) were used to fractionate cytosolic and nuclear proteins. Cyclohexamide (Sigma-Aldrich, St. Louis, MO) was reconstituted in DMSO. RNA extraction, reverse transcription, and real-time PCR Total RNA was extracted using TRIzol reagent, and converted to cDNA using SuperScript III First-Strand Synthesis kit (Invitrogen). Full length CDC25A cDNA was amplified using iProof High-Fidelity DNA polymerase (Bio-Rad, Hercules, CA), and cloned into pEF6/V5 His (Invitrogen, Carlsbad, CA) or pEGFP-N1(Clontech laboratories, CA) for standardization real time PCR reactions, UV radiation, CHX treatments, sequence analysis and restriction enzyme digestion, pEGFP-N1 fused to EGFP or m-Cherry was used for imaging and flowcytometry analysis or where indicated All DNA manipulations were performed in Biosafety level 1 or 2 laboratories. To quantify the total CDC25A transcript, real-time PCR assay were assembled using the forward primer 59- GCTCCTCCGAGTCAACAGAT -39, reverse primer 59- TGGACTACATCCCAACAGCTT-39, and FAMTM dye–labeled probe 59-ATTCTCCTGGGCCATTGGACA-39; to quantify the wild type CDC25A transcript, the assay was assembled using the forward primer 59 -GCTCCTCCGAGTCAACAGAT -39, re- verse primer 59- ACTACATCCCAACAGCTTCTG- 39 and FAMTM dye–labeled probe 59-ATTCTCCTGGGCCATTG- GACA-39. The assay was run in triplicate with TaqMan Fast Universal PCR master mix (Applied Biosystem, Carlsbad, CA) in Applied Biosystem 7900HT Fast Real Time PCR system. In all CDC25A-Q110del Novel Isoform Role in Lung Cancer reactions, GAPDH Fast TaqMan assay VIC dye–labeled probe was added as RNA loading control. When the total expression of CDC25A is designated as the endogenous reference gene, the abundance of CDC25AQ110del can be calculated as DCt = Ct wt2Ct tot. Hence, the relative abundance of CDC25wt in paired tumor versus normal tissue is calculated by 22DDCt method, where DDCt = DctTumor- DctNor- mal (User Bulletin #2 Applied Biosystem). If the expression levels of CDC25AQ110del and CDC25wt are equal in the corresponding tumor and the adjacent normal lung tissue, the calculated relative abundance value will be 1. A value,1 indicates that the tumor expresses a higher level of CDC25AQ110del than the paired normal lung tissue. Conversely, a value.1 indicates that the normal lung tissue expresses a higher level of CDC25AQ110del than the paired tumor. Sequence analysis and restriction enzyme digestion DNA clones were sequenced at the University of Maryland Baltimore sequencing facility or Genewiz Inc., (South Plainfield, NJ). Alignment was performed against CDC25A reference NM_001789. The cDNA clones used for the functional assays were amplified from NSCLC cell lines (Table S1). For enzymatic digestion analysis, a 292 bp fragment of CDC25A cDNA was amplified using primers: forward 59-CACTGGAGGTGAAGAA- CAACAG-39 and reverse 59-CAGCCACGAGATA- CAGGTCTTA-39, digested with the restriction endonuclease Bpu10I (New England Biolabs, Ipswich, MA) then separated on agarose gel. UV irradiation For UV treatment of cultured cells, the media was removed, cells were washed twice with phosphate-buffered saline and then irradiated in uncovered tissue culture dishes with 254 nm UV light (UVC) in Stratalinker UV Crosslinker (Stratagene, La Jolla, CA). Fresh culture medium was added back, and cells were further incubated for the described time points. Cell cycle analysis Cells were harvested, fixed in 70% ethanol, suspended in PI/ RNase staining Buffer (BD PharmingenTM, San Jose, CA) containing 0.1% Sodium Citrate and 0.1% Triton X-100. Data analysis was done in University of Maryland Baltimore Medical Center, FlowCytometry Core and analyzed with FlowJo software. Imaging analysis Cells expressing CDC25Awt or CDC25AQ110del- fused with mCherry or EGFP were fixed in 2% paraformaldehyde, permeabilized with 0.5% tritonX-100, stained with DAPI. The Images were captured using a Zeiss LSM 510 Meta Laser Scanning Confocal Microscope with DAPI, FITC and Rhoda- mine fiters. The histogram representative of the FITC and rhodamine expression was produced using image J software. Cell viability assay Cell viability was measured using the Cell Proliferation Reagent WST-1 (Roche Diagnostics Corporation, Indianapolis, IN). Patients and tissues Primary NSCLC tumors and their corresponding nonmalignant adjacent lung tissues from 88 individuals with pathologic stage I to IIIa NSCLC were evaluated. All of the patients were treated with surgery alone except those with stage IIIa disease who might also had received postoperative radiation therapy and adjuvant PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46464