The p16 protein is a cyclin-dependent kinase (CDK) inhibitor, which plays an important role in the regulation of the cell cycle by inactivating the cyclin-dependent kinase (CDK) that phosphorylates the retinoblastoma (Rb) protein. Overexpression of p16 protein has been found in many types of human malignancy. Autoantibody response to p16 in cancer has not been reported. This study determined the extent and frequency of autoantibodies to p16 in diverse malignancies. p16 recombinant protein was expressed in E. Coli BL21 (DE3) cells, and purified using GST fusion protein purification system. In further studies, p16 recombinant proteins were used as antigens in enzyme-linked immunoassay (ELISA) and Western blotting. Sera from 479 cancer patients and 82 normal individuals were analyzed. Autoantibodies to p16 were found in 11.7% in cancer, with significant difference from the normal individuals (p<0.05). The results in this study also showed that the frequency of antibodies to p16 is relatively higher in nasopharyngeal cancer (28.6%), breast cancer (17.1%) and hepatocellular carcinoma (HCC, 21.4%). Of the 56 ELISA positive sera with the anti-p16 antibodies, 85.7% (48/56) had positive reactions in Western blotting. The antigen-antibody absorption experiment was also performed to confirm the specificity of the anti-p16 antibody. In order to increase the frequency of antibody detection in cancer, a combination of three tumor-associated antigens (TAAs) p16, p53 and c-myc were used. Increased frequencies at p<0.01 were found for antibodies to p16 in breast, esophageal, and nasopharyngeal cancer as well as HCC. For antibodies to c-myc, increased frequencies at p<0.01 were found in breast, cervical, colorectal and lung cancer. For antibodies to p53, increased frequencies at p<0.01 were only found in breast cancer. With the successive addition of three TAAs, there was a stepwise increase of positive anti-body reaction up to 44% in breast cancer and 43% in nasopharyngeal cancer. In summary, the results in this study suggest that the combination of antibodies might acquire higher sensitivity for early cancer diagnosis. It is conceivable that auto-antibody profiles involving different panels or arrays of TAAs might be developed in the future and the results could be useful for cancer diagnosis.

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