Humoral immune response to p16, a cyclin-dependent kinase inhibitor in human malignancies

Abstract

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 phosphorylate retinoblastoma (Rb) protein. Overexpression of p16 protein has been found in many types of human malignancy. Antibody response to p16 in cancer has not been reported yet. This study determined the extent and frequency of autoantibodies to p16 in diverse malignancies. Cellular mRNA was extracted from Human epitheloid cervical carcinoma cells (Hela cell line), was further used as template for p16 cDNA cloning by Reverse-Transcriptase Polymerase Chain Reactions (RT-PCR). p16 cDNA sequence was subcloned into a pGEX expression vector which has a glutathione S-transferase (GST) gene, and a pGEX-p16 construct was made for subsequent p16 recombinant protein expression. p16 recombinant protein was expressed in E. Coli XL1-blue 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. (Abstract shortened by UMI.