CDC25A-Q110del Novel Isoform Role in Lung Cancer Figure 4. Cellular localization and mitotic activity of CDC25AQ110del. A. Immunoblot of H1299 cells showed CDC25AQ110del to localize more in the nucleus compared to the CDC25Awt. B. 24 hrs after UV irradiation, CDC25AQ110del showed more stability in H1299 and corresponded with more phosphorylation of DDR marker Chk1-ser345. C. Confocal Microscopy of 293F cells after 24 hr of transfection: CDC25AQ110del expression showed frequent mitotic figures (metaphase ‘‘thin arrows’’). Co-expression of CDC25Awt and CDC25AQ110del at (1:1) ratio, mitotic activity was still noticed (cytokinesis, ‘‘bold arrows’’). The histogram is representative of the pixel count for the FITC per condition and the rhodamine. D. pCDK1-Tyr15 dephosphorylation as downstream reader for relative increased phosphatase activity of CDC25AQ110del compared to CDC25Awt after 24 hr of transfection in 293F cells. doi:10.1371/journal.pone.0046464.g004 be needed to determine whether tumors with higher CDC25AQ110del are more resistant to DNA damaging agents or radiation therapy and if targeting CDC25AQ110del will sensitize these tumors to these treatments [44]. An interesting observation is the expression of CDC25AQ110del in the normal appearing lung tissues adjacent to NSCLC, sometimes with high abundance, suggesting the abnormality is an early event during lung carcinogenesis [44]. Although our study cannot determine whether CDC25AQ110del is also expressed in truly normal lung tissue or that its expression in the lung tissues evaluated is a result of the microscopic contamination with cancer cells from the adjacent tumor, several lines of evidence support our notion. First, 3 of the 4 immortalized human bronchial epithelial cell lines derived from patients with lung cancer expressed low level of CDC25AQ110del (Fig. 2C), indicating at least some normal bronchial epithelial cells may express CDC25AQ110del and its expression is independent of the presence of lung cancer cells. Second, the abundance of CDC25AQ110del relative to the total CDC25A is highly variable, from undetectable to almost 100%, suggesting the expression of CDC25AQ110del is determined by complicated genetic or environment factors that may also induce its expression in normal lung tissues. Third, there was no correlation of CDC25AQ110del expression levels between the NSCLC tumors and the paired adjacent normal lung tissues, suggesting the expression of CDC25AQ110del in the normal lung tissues was independent of the primary tumors. However, we did observe a relationship between higher CDC25AQ110del expression levels in the primary tumors and poor overall survival of the patients, particularly if the expression was significantly higher in the tumor compared to the paired adjacent lung tissue. The results suggest that CDC25AQ110del is an adverse factor in lung cancer progression or treatment response. Additional studies will be required to validate the findings and further explore biological functions of CDC25AQ110del in lung cancer initiation and progression. Supporting Information Figure S1 Identification of CDC25AQ110del in cDNA pool of NSCLC cell lines and tumor tissue. A. CDC25A RT-PCR product size: 292. Bpu10I restriction enzyme recognition sequence 59-CCˇ TNAGC-39 flanks the deletion site in CDC25AQ110del and cuts at 326 but not in the CDC25Awt. NEB digestion engine. B. Agarose gel shows Bpu10I digestion product of CDC25A amplified from NSCLC cell lines (lanes 2–5), and tumor tissue (lanes 8–12) using Bpu10I restriction endonuclease enzyme. Restriction fragment of CDC25AQ110del versus CDC25Awt clones used as control (lanes 6–7). Restriction fragment similar to that of the CDC25AQ110del clone digestion was noticed in the NSCLC cell lines and tumor tissue samples. (TIF) Figure S2 Increased accumulation of CDC25AQ110del protein compared to CDC25Awt. A. Fluorescent microscopy 72 hrs post transfection of 293F cells with CDC25AQ110del- mcherry versus CDC25Awt-mcherry showed prominent nuclear accumulation of CDC25AQ110del versus CDC25Awt. B. H1299 72 hrs after co-transfection with CDC25AQ110del and CDC25Awt, tagged with EGFP and mcherry fluorescent proteins alternatively. Figure 5. Clinical Significance of CDC25AQ110del. A. Kaplan Meier survival curves showed CDC25AQ110del in tumor tissue to correlate with poor overall survival of NSCLC patients (log rank P = .074). B. Kaplan Meier Survival curves showed that when CDC25Awt is higher in tumor versus normal tissue pair, it correlated with better overall survival (log rank P = .0018). Relative Quantification of target gene ‘‘CDC25Awt’’ in NSCLC tumor tissue relative to normal tissue pair of NSCLC patients according to the formula: 22DDct. CDC25A template of tumor or normal was run in triplicates of uniplex reaction for each of the Ctwt and Cttot assays, and the mean was calculated for each assay. doi:10.1371/journal.pone.0046464.g005 PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e46464