CDC25A-Q110del Novel Isoform Role in Lung Cancer and S124). E. Schematic illustration of suggested alternative splicing site. Alternative donor site: a 59 splice junction (donor site), changing the 39 boundary of the upstream exon, would produce the CDC25Awt transcript, while an Alternative acceptor site: a 39 splice junction (acceptor site), changing the 59 boundary of the downstream exon, would produce the CDC25AQ110del transcript. doi:10.1371/journal.pone.0046464.g001 chemotherapy, at the University of Texas M. D. Anderson Cancer Center (MDACC) from 1995 to 2000. Samples were immediately frozen and stored at 280uC. The selection of these patients was based on the availability of archived fresh tumor and correspond- ing normal lung tissues for the investigators. Clinical information and follow-up information for the study were based on chart review and form reports from MDACC tumor registry service. Informed consent for the use of residual resected tissues for research was obtained from all the patients enrolled in the study. Ethics statement Written informed consent to use residual resected tissue for research was obtained from all patients enrolled in the study. The consent procedure and the use of these material and clinical information was reviewed and approved by University of Texas MDACC surveillance committee. Statistical analysis Student t-test was used for statistical analysis of the functional assays. Survival analysis Kaplan Meier curves were generated with log rank analysis utilizing Proc Lifetest in SAS 9.2. All tests are two-sided and P values,.05 is considered statistically significant. Results Identification of CDC25AQ110del in NSCLC To investigate potential alterations of CDC25A at mRNA level, we sequenced CDC25A cDNA clones derived from a panel of 10 NSCLC cell lines. Among total 16 cDNA clones from the 10 cell lines, we observed a specific trinucleotide deletion in 7 of the 16 clones from 5 of the 10 cell lines (Fig. 1A) (Table S1). The deletion locates at positions 328–330 in reference to NM_001789.2, CDC25A transcript 1, which predicts a glutamine deletion at codon 110 (Fig. 1B). This amino acid residue is situated within the regulatory domain of CDC25A, and is conserved among several vertebrates (Fig. 1C and D). We term the novel CDC25A isoform with codon 110 deletion as CDC25AQ110del. This deletion is likely a result of alternative RNA splicing, since no alteration of genomic DNA sequence were found in the NSCLC cell lines (data not shown) (Fig. 1E) To confirm the presence of CDC25AQ110del in NSCLC cell lines and primary NSCLC tumor tissues, we examined cDNAs from 4 NSCLC cell lines and 5 primary NSCLC tumor tissues using restriction endonuclease digestion by Bpu10I, which can cleave the sequence 59-CCTNAGC, a unique site in CDC25AQ110del sequence, to produce a shorter cleaved DNA band. All the samples showed the shorter cleaved DNA band at various densities (Fig. S1). We next devised a real-time PCR assay (Fig. 2A) to assess the quantity of CDC25AQ110del among the total CDC25A transcripts in NSCLC cell lines and tissue samples, to demonstrate that the assay can quantitatively measure the relative abundance of CDC25A isoforms, we constructed a Ct curve using purified plasmid DNA containing either CDC25Awt or CDC25AQ110del cDNA insert. The result showed a nearly linear relationship with different wild type and Q110del ratio (Fig. 2B).This method was then used to asses CDC25AQ110del expression in cell lines and tissues. In 4 HBEC cell lines, CDC25AQ110del expression was detectable but at generally less than 20% of the total CDC25A transcripts (Fig. 2C). It should be noted that these cell lines were derived from immortalized human bronchial epithelial cells of patients with lung cancer. In comparison, 9 (75%) of the 12 NSCLC cell lines expressed CDC25AQ110del greater than 20% of the total CDC25A transcripts including 3 (20%) of the cell lines expressed CDC25AQ110del at almost 50% level (Fig. 2C). The difference of CDC25AQ110del expression levels between the HBEC cell lines and the NSCLC cell lines was statistically significant (P = .003). Interestingly, the H596 and the H549 cell lines that show CDC25A Q110del at .50% of total CDC25A, has a high modal chromosomal number at a high percentage, NCI-H596 modal number = 71; range = 65 to 75.This is a near triploid human cell line. While A549 is a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells (according to ‘‘American Type Culture Collection’’). This suggests CDC25AQ110del to play a role in genomic instability and cumulative malignant changes [26]. A correlation between CDC25AQ110deland accumulation of hyperploid cells is described below in cell cycle distribution. CDC25AQ110del protein stability and Cell survival CDC25A is a labile protein, tightly regulated through several phosphorylation events in its regulatory domain [8]. To investigate the effect of Q110 deletion on the fate of CDC25A protein regulation, we tested the degradation rate of CDC25AQ110del using cyclohexamide (CHX) treatment. In H1299 cells treated with CHX, we observed that CDC25AQ110del has a longer half-life compared to CDC25Awt (Fig. 3A). Furthermore, when CDC25Awt and CDC25AQ110del were co-transfected, more CDC25AQ110del was accumulated than CDC25Awt at 72 h after transfection (Fig. S2) in both H1299 and 293F cells. In 293F cells transfected with either CDC25Awt or CDC25AQ110del and treated with 10 and 20 j/m2 UV radiation, cells transfected with CDC25AQ110del showed an enhanced protein stability 30 min after the UV irradiation but not the cells transfected with CDC25Awt (Fig. 3B). To get a more quantitative measurement of CDC25AQ110del and CDC25Awt levels, we measured the fluorescent intensity of CDC25A-EGFP fusion proteins gating equal number of 293F cells expressing CDC25Awt-EGFP or CDC25AQ110del-EGFP and observed a significantly higher level of fluorescent intensity in the CDC25AQ110del-EGFP transfected cells (Fig. 3C). The cell cycle analysis of the same gated population of cells, showed increased post G2 population (hyperploid cells) of the CDC25Awt- EGFP expressing cells compared to the CDC25AQ110del-EGFP, while the CDC25AQ110del-EGFP accelerated the cells more through the post G2 phase (mitosis) compared to the CDC25Awt (p = 0.0047) (Fig. 3D). This suggests that the CDC25AQ110del can abrogate the G2/M check point compared to the CDC25Awt, driving the cells more through mitosis [26,27]. To investigate if the CDC25AQ110del can affect the survival of NSCLC cells under perturbed conditions, H1299 cells transfected with CDC25AQ110del were treated with UV radiation at different doses, H1299 expressing CDC25AQ110del were more resistant to UV induced cell death compared to the cells transfected with the control vector or CDC25Awt, particularly at high UV doses (Fig. 3E). PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46464