However the tumor-node-metastasis (TNM) system is helpful for this purpose,2 it is not usually correlated with outcome of the cancer patients. regression models revealed that ARD1A-positive patients had significantly shortened overall survival (OS) Raltegravir (MK-0518) (HR, 1.91, = 0.039) Raltegravir (MK-0518) and borderline significantly shortened disease-free survival (DFS) (HR, 1.70; = 0.068). KaplanCMeier survival curves also showed that ARD1A expression was associated significantly with shortened DFS Raltegravir (MK-0518) (= 0.037) and OS (= 0.019). These results indicate that ARD1A is usually a novel tumor-associated antigen and a potential prognostic factor for colon cancer. Colon cancer is one of the most common cancers worldwide and is the second leading cause of cancer death in developed countries.1 The 5-12 months survival rate of patients with colon cancer has improved during the past decade because of advances in screening and systemic treatment, but the survival rate remains relatively poor in patients with stage III and IV cancer. 2 Colon cancer is usually treated mainly by surgery, and it would be beneficial if the prognosis after initial surgery could be predicted. Even though tumor-node-metastasis (TNM) system is helpful for this purpose,2 it is not usually correlated with end result of the malignancy patients. To predict the prognosis more exactly, new molecular markers are needed to be exploited. Considerable evidence has shown that an immune response in the form of autoantibodies to numerous tumor antigens was developed in patients with malignancy.3,4,5,6,7 Thus, autoantibodies in the serum of patients with colon cancer could be used to isolate specific tumor-associated antigens. More sensitive and specific molecular markers have the potential to improve the preclinical diagnosis of main and recurrent colon cancer, as well as hold the promise of prognostic value. Phage display libraries have proved to be a useful tool for identifying autoantigens or epitopes recognized by antibodies.8,9,10,11,12 Selection of peptide libraries with mAbs and mixed serum from patients with disease has led to the isolation of immunoreactive peptide epitopes.4,12,13,14 The strategy has also led to the identification of some tumor-associated antigens from phage display libraries using patient serum.4,12 In the present study, by screening a phage display peptide library with serum antibodies from patients with colon cancer, together with bioinformatic analysis and a series of immune experiments, we first revealed that human arrest defective 1 (ARD1A) may serve as an indication of unfavorable prognosis in colon cancer. Materials and Methods Cell Collection and Cell Culture Conditions The LoVo colon adenocarcinoma cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). The LoVo cells were produced in DMEM Rabbit polyclonal to Caspase 8.This gene encodes a protein that is a member of the cysteine-aspartic acid protease (caspase) family.Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. (Gibco) product with 10% fetal bovine serum (FBS, PAA Laboratories, Pasching, Austria) Raltegravir (MK-0518) and antibiotics (100 models/ml penicillin and 100 g/ml streptomycin) at 37C in a humidified incubator with 5% CO2. Peptide Screening and Phage ELISA Sera from five patients with colon cancer before any therapeutic intervention were obtained from Tissue Lender of Peking University or college School of Oncology. The clinicopathological features and TNM staging are summarized in Supplemental Table S1 at strain ER2738 grown to the log phase. Amplified phages were used in the next cycle. After four rounds of selection, 1000 plaques were picked up randomly and analyzed for reactivity with serum from colon cancer patients using phage ELISA. Amplified phages from a single clone were added to the wells of a microtiter plate precoated with a 1:200 dilution of serum from colon cancer patients or healthy volunteers in 0.1 M of bicarbonate buffer (pH 9.5) and then blocked with 5% skim milk in PBS. The plate was incubated at room temperature for 1 hour and washed five occasions with 0.05% Tween-20/PBS. Bound phages were detected by incubation with HRP-conjugated anti-M13 antibody (Amersham Biosciences, Piscataway, NJ) for 1 hour, followed by washing and addition of a peroxidase substrate (values are two-sided, and 0.05 was considered statistically significant. Results Peptides Specially Reacted with Serum from Colon Cancer Patients Were Recognized We used a random 12-mer phage.